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Chapter 10 Naturally Occurring Modified Nucleosides in DNA
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CHAPTER 10 NATURALLY OCCURRING MODIFIED NUCLEOSIDES I N DNA MELANIE EHRLICH and XIAN-YANG ZHANG Department o f Biochemistry, Tulane Nedical School, New Orleans, LA 70112, U . S . A .
TABLE 10.1 10.2 10.3
10.4 10.5
10.6 10.7 10.8
OF CONTENTS Introduction H i g h l y M o d i f i e d Bacteriophage DNA M o d i f i e d Bases i n DNA f r o m B a c t e r i a and Lower Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . Meth l a t i o n o f t h e DNA o f M i t o c h o n d r i a , C h l o r o p l a s t s , and h k a r y o t i c V i r u s e s D i s t r i b u t i o n o f m5C i n t h e N u c l e a r DNA o f H i g h e r P l a n t s and V e r t e b r a t e s The F u n c t i o n a l S i g n i f i c a n c e o f V e r t e b r a t e DNA Meth 1a t i o n : T r a n s c r i p t i o n , Chromatin S t r u c t u r e , DNA R e p r i c a t i o n and Repair, Cancer and Embryogenesis Summary References
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INTRODUCTION To s t a t e t h a t DNA i s composed o f o n l y f o u r bases, adenine (A) t thymine (T), guanine (G) and c y t o s i n e (C) i s an o v e r - s i m p l i f i c a t i o n because most t y p e s o f DNA n a t u r a l l y c o n t a i n one t o t h r e e a d d i t i o n a l bases, m o d i f i e d forms o f A o r C. U s u a l l y , o n l y a m i n o r The p o r t i o n o f t h e C o r A r e s i d u e s i s m o d i f i e d ( r e f s . 1-3). e x c e p t i o n s a r e t h e DNA from c e r t a i n b a c t e r i o p h a g e s ( r e f . 4) and from some d i n o f l a g e l l a t e s ( r e f . 5 ) . One o r a n o t h e r o f a v a r i e t y o f m o d i f i e d p y r i m i d i n e s a r e found i n t h e s e phage DNAs and 5h y d r o x y m e t h y l u r a c i l i n d i n o f l a g e l l a t e DNA. I n c o n t r a s t , t h e o n l y m o d i f i e d bases found i n a1 1 o t h e r DNAs a r e 5-methyl cytosine (rn5C), N4-methylcytosine (m4C), and N6-methyladenine (m6A). The f u n c t i o n s o f t h e s e DNA m o d i f i c a t i o n s a r e d i v e r s e and range f r o m p r o t e c t i o n o f b a c t e r i a l h o s t DNA a g a i n s t a pathway f o r d e g r a d i n g f o r e i g n DNA t o c o n t r o l o f p r o k a r y o t i c and e u k a r y o t i c t r a n s c r i p t i o n a l a c t i v i t y .
10.1
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HIGHLY MODIFIED BACTERIOPHAGE DNA Some t y p e s of b a c t e r i a l viruses, although not most, have one of the DNA bases completely o r l a r g e l y replaced by a modified derivative (ref. 4). These highly modified phage DNAs almost always c o n t a i n pyrimidine m o d i f i c a t i o n s . The f i r s t of these t o be di scovered was 5-hydroxymethyl c y t o s i ne (hm5C) i n phage T2, T4, and T6 DNAs ( r e f . 6 ) . These e n t e r i c Escherichia c o y i phages, which were among the e a r l i e s t s u b j e c t s of i n t e n s i v e molecular b i o l o g i c a l i n v e s t i g a t i o n , a r e very highly modified, a f a c t which sometimes complicated t h e i r use a s experimental models. Not only do they have a hydroxymethyl group a t each C r e s i d u e , b u t a l s o , 75-100% o f t h e i r r e s u l t i ng hm5C r e s i d u e s a r e gl ucosyl a t e d ( r e f s . 7-9). The type of l i n k a g e of t h e glucose and t h e number of g l u c o s e m o i e t i e s per hm5C r e s i d u e depends upon t h e phage type ( r e f . 1 0 ) . In T2 phage DNA, 70% of t h e hm5C r e s i d u e s c o n t a i n an a-glucosyl moiety, 5% have a g e n t i o b i o s e (8-91 ucosyl -a-gl ucosyl) , and the rest a r e nonglucosylated. Phage T4 and T6 DNAs have the f o l l o w i n g d i s t r i b u t i o n s of t h e i r hm5C r e s i d u e s : T4, 70% a - g l u c o s y l a t e d , 30%-8glucosyl a t e d , and 0% nongl ucosyl a t e d ; T6, 3% 0-91 ucosyl a t e d , 72% 8-1,6-91 ucosyl -a-gl ucosyl a t e d ( g e n t i o b i o s e ) , and 25% nongl ucosyla t e d . The formation of t h e hydroxymethyl group i s c a t a l y z e d a t t h e deoxymononucl e o t i d e 1 eve1 by a phage-encoded dCMP hydroxymethylt r a n s f e r a s e p r i o r t o i n c o r p o r a t i o n i n t o the DNA ( r e f . 1 1 ) . The s y n t h e s i s of this phage DNA e x p l o i t s t h e h o s t ' s r e s o u r c e s by degrading h o s t DNA and dCTP and converting t h e r e s u l t i n g recycled dCMP a s well a s de nova synthesized dCMP t o hm5dCTP. This phosphorylation involves a phage-encoded deoxynucleoside monophosphate kinase and a host diphosphate kinase a s well a s the phage hydroxymethyl t r a n s f e r a s e ( r e f . 1 1 ) . A f t e r i n c o r p o r a t i o n of the hm5dCMP r e s i d u e s , the gl ucosyl a t i o n of t h e hydroxymethyl groups i nvol ves hmT-speci f i c a- and 8-gl ucosyl t r a n s f e r a s e s , which have some sequence s p e c i f i c i t y ( r e f . 1 2 ) . This g l u c o s y l a t i o n i s normally necessary t o prevent t h e degradation of these phage DNAs by E . c o l i nucleases a s well a s by phage-encoded n u c l e a s e s meant t o d e s t r o y h o s t DNA ( r e f s . 13-16). I f dCMP r e s i d u e s a r e i n c o r p o r a t e d i n t o phage DNA, the DNA w i l l be degraded by the T4-encoded endonuclease I 1 ( r e f . 1 6 ) . Therefore, hydroxymethyl a t i o n and glucos y l a t i o n of c y t o s i n e r e s i d u e s i n T-even phage p l a y an important 10.2
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r o l e i n t h e phage l i f e c y c l e by p r o t e c t i n g phage DNA f r o m degradat i o n w h i l e a l l o w i n g h o s t DNA t o be s p e c i f i c a l l y r e c o g n i z e d f o r such h y d r o l y s i s . I t a l s o f a c i l i t a t e s p h a g e - s p e c i f i c t r a n s c r i p t i o n l a t e i n i n f e c t i o n c a t a l y z e d by a phage-modified h o s t RNA p o l y merase ( r e f s . 4, 17, 18). L i k e many o t h e r types o f b a c t e r i o p h a g e DNAs i n c l u d i n g t h o s e which a r e n o t h i g h l y m o d i f i e d ( r e f s . 19, 20), T4 DNA c o n t a i n s m i n o r amounts o f m6A ( r e f . 21). T h i s m e t h y l a t i o n o f A r e s i d u e s i s c a t a l y z e d by a T4-induced, sequence-specific DNA methyl t r a n s f e r a s e ( r e f . 21). The p h y s i o l o g i c a l s i g n i f i c a n c e o f m i n o r amounts o f m6A o r m5C i n phage DNAs remains t o be d e t e r mined. E x t e n s i v e hydroxymethyl a t i o n occurs a t a n o t h e r p y r i m i d i n e r e s i d u e i n a s e t o f c l o s e l y r e l a t e d phage DNAs which a r e widespread i n s o i l ( r e f . 22). A few p a r t i a l l y homologous phages t h a t i n f e c t Bacillus subtilis, SPO1, ue, SP8, SP82G, 2C, and SP5, have t h e T r e s i d u e s i n t h e i r genomes c o m p l e t e l y r e p l a c e d by 5-hydroxym e t h y l u r a c i l (hm5U) r e s i d u e s ( r e f s . 11, 22) which a r e nonglucosy1 ated. L i k e t h e T-even phages, t h e s e phages hydroxymethylate a t t h e mononucleotide l e v e l , i n t h i s case, w i t h dUMP as t h e s u b s t r a t e . The r e a c t i o n i s s i m i l a r l y c a t a l y z e d by a phage-induced hydroxymethylase ( r e f . 23). To p r e v e n t i n c o r p o r a t i o n of dTTP, t h e phages encode a dTTPase ( r e f . 24) as w e l l as an i n h i b i t o r o f t h e h o s t b a c t e r i u m ' s t h y m i d y l a t e s y n t h e t a s e ( r e f . 2 3 ) . Channeling of p y r i m i d i n e s t o s y n t h e s i s o f hm5dUTP i s f a c i l i t a t e d by two phageinduced enzymes, a dCMP deaminase and a deoxynucleoside monophosphate k i n a s e ( r e f s . 4, 23). I n h i b i t i o n o f h o s t DNA r e p l i c a t i o n occurs a f t e r i n f e c t i o n i n i t i a l l y as t h e r e s u l t o f a phage-induced i n h i b i t o r o t h e r than t h e i n h i b i t o r o f t h y m i d y l a t e s y n t h e t a s e ( r e f . 22). U n l i k e t h e case f o r t h e T-even phages, h o s t DNA i s n o t e x t e n s i v e l y degraded and t h e hm5U-containing B . s u b t i l i s phages can have as much as 20% o f t h e i r hm5U r e p l a c e d by T w i t h o u t l o s i n g t h e i r v i a b i l i t y ( r e f . 25). Nonetheless, t r a n s c r i p t i o n i n t h e m i d d l e and l a t e stages o f t h e i n f e c t i o u s c y c l e r e q u i r e s hm*Uc o n t a i n i n g phage DNA sequences as w e l l as a phage-encoded regul a t o r y p r o t e i n r e p l a c i n g one of t h e h o s t RNA polymerase's sigma f a c t o r s ( r e f . 26). I n a d d i t i o n , a DNA-binding p r o t e i n which b i n d s s p e c i f i c a l l y t o c e r t a i n hm5U-containing sequences i s s y n t h e s i z e d ( r e f . 27).
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Another t y p e o f B. subtilis phage has a d i f f e r e n t s u b s t i t u e n t r e p l a c i n g t h e T r e s i d u e s of i t s DNA, namely u r a c i l (U) r e s i d u e s . J u s t as T r e s i d u e s a r e commonly found i n RNA ( t R N A ) a l t h o u g h t h e y a r e m o s t l y a s s o c i a t e d w i t h DNA, so U r e s i d u e s can be a normal c o n s t i t u e n t o f DNA. To date, g e n e t i c a l l y programmed f o r m a t i o n o f U r e s i d u e s i n DNA has been found o n l y i n phage PBSl and i t s v e r y c l o s e l y r e l a t e d d e r i v a t i v e PBS2 ( r e f s . 28, 29). PBS2 has been shown t o encode a dTMP 5'-phosphatase, which h e l p s p r e v e n t T r e s i d u e s from b e i n g i n c o r p o r a t e d i n t o phage DNA ( r e f . 30), and a dUMP kinase, which t o g e t h e r w i t h a h o s t n u c l e o s i d e diphosphate k i n a s e p r o v i d e s dUTP f o r phage DNA s y n t h e s i s . The phage d u r i n g i t s l i f e c y c l e has t o cope w i t h h o s t mechanisms f o r e x c l u d i n g advent i t i o u s U r e s i d u e s from DNA, namely, a h o s t dUTPase and a h o s t uracil-DNA g l y c o s y l a s e ( r e f . 31). The l a t t e r enzyme e x c i s e s t h e n o r m a l l y small amount o f m i s i n c o r p o r a t e d dUTP (dUMP r e s i d u e s ) as w e l l as U r e s i d u e s a r i s i n g from spontaneously o c c u r r i n g heatinduced deamination o f C r e s i d u e s ( r e f s . 32, 33). To c o u n t e r a c t t h e a c t i v i t y o f t h e s e enzymes, phage i n f e c t i o n induces t h e synt h e s i s o f i n h i b i t o r s o f t h e DNA-uracil e x c i s i o n pathway and o f dUTPase ( r e f s . 31, 34, 35). Another phage t h a t i n f e c t s B. subtilis has a c o m p l i c a t e d and unique t y p e o f m o d i f i c a t i o n i n i t s DNA. Phage SP15 DNA c o n t a i n s 60% o f i t s T r e s i d u e s r e p l a c e d by 5 - ( 4 ' ,5'-dihydroxypenty1)uracil (hpsU) ( r e f . 36) i n which a f i v e - c a r b o n s i d e c h a i n w i t h two hydroxyl groups i s bonded t o t h e 5 p o s i t i o n o f t h e p y r i m i d i n e , t h e same p o s i t i o n m o d i f i e d i n a l l t h e above-mentioned p y r i m i d i n e s . Attached t o one o f t h e h y d r o x y l groups i s a glucose m o e i t y and t o t h e o t h e r v i a a phosphodiester l i n k a g e i s a phosphoglucuronate ( r e f . 37). T h i s g l u c u r o n i c acid-l-phosphate m o i e t y i n t r o d u c e s a phosphate t h a t i s n o t p a r t o f t h e DNA's phosphodiester backbone. I t c o n f e r s e x t r a n e g a t i v e changes on t h e DNA and i s a p p a r e n t l y responsi b l e f o r t h e a1 k a l ine 1a b i 1i t y and 1ow me1ti ng temperature o f t h i s DNA ( r e f . 38). SP15 DNA i s t h e o n l y known DNA s t a b l y c o n t a i n i n g phosphates t h a t a r e n o t p a r t o f t h e phosphodiester backbone. The d i s t r i b u t i o n o f hp5U versus T r e s i d u e s i n SP15 DNA i s .nonrandom. How hp5U, which o n l y partially r e p l a c e s T i n t h i s DNA, i s i n t r o d u c e d i n t o s p e c i f i c p o s i t i o n s remains unknown ( r e f . 37). Less w e l l d e f i n e d c h e m i c a l l y i s t h e h y p e r m o d i f i e d T d e r i v a -
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t i v e which p a r t i a l l y r e p l a c e s T i n t h e B. subtilis phage S P l O DNA. The s y n t h e s i s o f t h i s DNA i n v o l v e s a novel pathway i n which hm5U r e s i d u e s ( f r o m hm5dUTP) a r e i n c o r p o r a t e d i n t o t h e DNA and t h e n is pyrophosphorylated. Subsequently, 15% of i t l o s e s t h e pyrophosphate and i s converted t o t h e hypermodified T r e s i d u e s and t h e r e s t i s converted t o T r e s i d u e s ( r e f . 39). A s i m i l a r pathway i s encoded by phage dW14 t h a t i n f e c t s Pseudomonas acidovorans. The DNA o f t h i s phage has h a l f o f i t s T r e s i d u e s r e p l aced by 5- (4-ami n o b u t y l ami nomethyl ) u r a c i 1 (a-putresc i n y l t h y m i n e ; putT) which c o n f e r s a p o s i t i v e charge on t h e r e s i d u e ( r e f . 4 ) . T h i s base i s r e s p o n s i b l e f o r t h e higher-than-expected m e l t i n g temperature o f t h e DNA and i t s a l k a l i l a b i l i t y ( r e f . 40). Both putT and T i n dW14 DNA a r i s e from hm5dUTP. A f t e r dW14 i n f e c t i o n , t h e h o s t s u f f e r s t h e gradual l o s s o f dTTP and t h e appearance o f hm5dUTP c a t a l y z e d by a phage-induced enzyme ( r e f . 41). T h i s hm5dUTP i s i n c o r p o r a t e d i n t o t h e DNA and, a t t h e p o l y n u c l e o t i d e l e v e l , pyrophosphorylated ( r e f . 42). As f o r t h e T r e s i d u e s o f SPlO, T r e s i d u e s i n dW14 a r e formed a t t h e DNA l e v e l f r o m 5[(hydroxymethyl)-0-pyrophosphoryl] u r a c i 1 r e s i d u e s ( r e f s . 41, 42). The d i s t r i b u t i o n o f putT, which o n l y p a r t i a l l y r e p l a c e s i t s corresponding unmodified m a j o r base, i s s e q u e n c e - s p e c i f i c ( r e f . 43) as i n t h e case of hp5U i n SP15 DNA. Two more examples o f unusual bases i n h i g h l y m o d i f i e d phage DNAs have been r e p o r t e d . One i s a C d e r i v a t i v e , 2,5-dihydroxy-4ami nopyrimi d i n e , (5-hydroxycytosi ne) , whi ch c o m p l e t e l y r e p l aces C i n t h e DNA o f phage N-17, whose h o s t i s Shigella flexneri ( r e f . 44). The o t h e r i s t h e o n l y r e p o r t e d example o f a h i g h l y m o d i f i e d DNA c o n t a i n i n g as a m a j o r base a p u r i n e d e r i v a t i v e . T h i s i s found i n cyanophage S-2L, which i n f e c t s t h e b l ue-green a l g a e S y n e c h o c o c c u s e l o n g a t u s . I t has been i d e n t i f i e d as 2-aminoadenine and c o m p l e t e l y r e p l a c e s A ( r e f . 45). The o n l y o t h e r example o f extens i v e , b u t n o t major, replacement o f a p u r i n e i n DNA i s found i n E . c o l i phage Mu DNA. T h i s phage encodes t h e c o n v e r s i o n o f 15% o f i t s A r e s i d u e s t o a-N-(9-~-0-2'-deoxyri b o f u r a n o s y l p u r i n-6-y1)glycinamide i n which an acetamido group i s on t h e N-6 o f adenine ( r e f . 46). A l l o f t h e above-mentioned phage DNAs have as t h e i r main m o d i f i e d base a d e r i v a t i v e n o t commonly found i n o t h e r DNAs. Only
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one o f t h e s e , hm5U, has been f o u n d i n any DNA o t h e r t h a n t h a t o f phage (see b e l o w ) . There i s one example o f a h i g h l y m o d i f i e d DNA, phage XP12 DNA, whose m o d i f i e d base i s a m a j o r component o f t h i s phage DNA and a m i n o r component o f many o t h e r DNAs. Phage XP12 was d i s c o v e r e d b y Tsong-Teh Kuo g r o w i n g on t h e p l a n t p a t h o g e n X a n t h o m o n a s oryzae ( r e f . 47). XP12 DNA has e s s e n t i a l l y a l l o f i t s C r e s i d u e s r e p 1 aced b y 5-methyl c y t o s i n e (m5C) ( r e f . 48-50). T h i s base i s a l s o f o u n d as a m i n o r component o f v e r y many t y p e s o f DNAs (see b e l o w ) . The m5C r e s i d u e s g i v e XP12 DNA a m e l t i n g t e m p e r a t u r e h i g h e r t h a n expected; t h e r e f o r e , t h e 5-methyl a t i o n i n c r e a s e s t h e s t a b i l i t y o f t h e h e l i x ( r e f . 49). The s o u r c e o f t h e m e t h y l g r o u p f o r XP12 D N A ' s m5C r e s i d u e s (and f o r T r e s i d u e s , i n g e n e r a l ) i s t h e 3-carbon o f s e r i n e r a t h e r t h a n t h e t h i o m e t h y l c a r b o n o f methi o n i n e as i n t h e f o r m a t i o n o f m5C as a m i n o r base i n DNA ( r e f . 49). M e t h y l a t i o n o f t h e C r e s i d u e s f o r XP12 DNA o c c u r s a t t h e m o n o n u c l e o t i d e l e v e l i n a r e a c t i o n c a t a l y z e d b y a phage-induced d e o x y c y t i d y l a t e m e t h y l t r a n s f e r a s e ( r e f . 51). XP12 i n f e c t i o n i n d u c e s t h e s y n t h e s i s o f a 5-methyldeoxyc y t i d i n e 5 ' - monophosphate k i n a s e , which, u n l i k e E . c o l i and X . oryzae m o n o n u c l e o t i d e k i n a s e s , can c a t a l y z e t h e p h o s p h o r y l a t i o n o f m5dCMP ( r e f . 52). The c o n v e r s i o n o f m5dCDP t o m5dCTP i s app a r e n t l y catalyzed by a r e l a t i v e l y n o n s p e c i f i c h o s t nucleoside d i p h o s p h a t e k i n a s e ( r e f . 52). I n t e r e s t i n g l y , XP12 u t i l i z e s t h e m5dCTP i n a u n i q u e pathway f o r p r o d u c t i o n o f T r e s i d u e s . I t i n d u c e s t h e f o r m a t i o n o f an mSdCTP deaminase t h a t g e n e r a t e s dTTP b y d e a m i n a t i o n o f p a r t o f t h e p o o l o f n e w l y c r e a t e d m5dCTP i n p h a g e - i n f e c t e d c e l l s ( r e f . 53). The phage a l s o i n d u c e s t h e synt h e s i s o f an e x o n u c l e a s e w i t h a p r e f e r e n c e f o r d o u b l e - s t r a n d e d DNA ( r e f . 5 4 ) . However, t h i s enzyme h y d r o l y z e s XP12 DNA and h o s t DNA e q u a l l y w e l l . T h i s c o n t r a s t s w i t h a h o s t e x o n u c l e a s e t h a t has a p r e f e r e n c e f o r s i n g l e - s t r a n d e d DNA and h y d r o l y z e s h o s t DNA much b e t t e r t h a n XP12 DNA ( r e f . 54, 55). Thus, some phage-induced enzymes i n v o l v e d i n m e t a b o l i s m o f h i g h l y m o d i f i e d phage DNAs may n o t have a s e l e c t i v i t y f o r t h e m o d i f i e d DNA, b u t r a t h e r may j u s t n o t be i n h i b i t e d by t h e m o d i f i c a t i o n . 10.3
MODIFIED BASES I N DNA FROM BACTERIA AND LOWER EUKARYOTES B a c t e r i a u s u a l l y c o n t a i n one o r more o f t h e f o l l o w i n g m e t h y l -
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ated bases i n t h e i r DNA: m6A, m5C, and t h e newly d i s c o v e r e d m4C ( r e f s . 3, 56-61). These m e t h y l a t e d bases u s u a l l y c o n s t i t u t e 0.010.7% o f t h e r e s i d u e s of t h e DNA, a l t h o u g h 2 mol% m6A i s p r e s e n t i n one b a c t e r i a l genome ( r e f s . 3, 61, 62). The r e l a t i v e frequency o f occurrence o f m i n o r amounts these m o d i f i e d bases i n b a c t e r i a l DNAs i s m6A > m5C > m4C ( r e f . 61). Also, i t i s o n l y m6A t h a t has been found a t l e v e l s > 0 . 4 mol% ( r e f s . 3, 61). These a r e t h e o n l y observed m o d i f i e d bases i n b a c t e r i a l DNA and a l t h o u g h t h e g r e a t m a j o r i t y o f s t u d i e d b a c t e r i a c o n t a i n a t l e a s t one o f them i n t h e i r genomes, some b a c t e r i a l DNAs have no d e t e c t a b l e m o d i f i e d bases ( r e f s . 3, 61). Much o f t h i s b a c t e r i a l DNA m e t h y l a t i o n i s p a r t o f r e s t r i c t i o n / m o d i f i c a t i o n systems ( r e f . 63). These i n v o l v e sequence-speci f i c adenine- o r c y t o s i ne-DNA methyl t r a n s f e r a s e s and corresponding sequence-speci f i c endonucl eases ( r e s t r i c t i o n endonucleases) R e s t r i c t i o n endonucleases general l y c l e a v e t h e same o l i g o n u c l e o t i d e recognized by t h e methyl t r a n s f e r a s e and w i 11 c l e a v e t h e sequence o n l y i n i t s unmethylated form. However, i n a t l e a s t one case, a r e s t r i c t i o n endonuclease c l e a v e s o n l y t h e a p p r o p r i a t e l y methylated r e c o g n i t i o n sequences r a t h e r than t h e unmethylated sequence ( r e f . 64). The restriction/modification systems s e r v e t o demarcate h o s t DNA as s e l f and a l l o w phage DNA which had i n f e c t e d a b a c t e r i a l s t r a i n w i t h a h e t e r o l o g o u s m o d i f i c a t i o n system o r o t h e r f o r e i g n DNA e n t e r i n g t h e b a c t e r i a l c e l l t o be recognized as n o n - s e l f and thereby, be degraded ( r e f s . 65, 66). However, t h e f u n c t i o n s o f t h e m o d i f i c a t i o n and r e s t r i c t i o n enzymes may not be l i m i t e d t o e x c l u s i o n o f f o r e i g n DNA. Also, some bact e r i a1 DNA methyl t r a n s f e r a s e s recogni ze s p e c i f i c DNA sequences f o r which no corresponding r e s t r i c t i o n enzyme e x i s t s i n t h e h o s t bacterium. The most w e l l s t u d i e d example o f t h e s e i s t h e DNA adenine m e t h y l a t i o n (dam) system o f E. coli which c o n v e r t s 5 ' GATC-3 ' sequences t o 5 ' -Gm6ATC-3 ' ( r e f . 67) Dam m e t h y l a t i o n d i r e c t s DNA mismatch r e p a i r as f o l l o w s ( r e f s . 68, 69). The r e c o g n i t i o n sequence i s a palindrome so t h a t n o r m a l l y b o t h s t r a n d s o f t h e DNA a r e s y m m e t r i c a l l y m e t h y l a t e d a t 5'-GATC-3' s i t e s . However, immediately a f t e r DNA s y n t h e s i s o n l y one s t r a n d , t h e t e m p l a t e s t r a n d , i s m e t h y l a t e d ( r e f s . 70, 71). T h i s hemim e t h y l a t i o n a l l o w s t h e nascent s t r a n d t o be d i s t i n g u i s h e d from t h e t e m p l a t e so t h a t t h e DNA mismatch r e p a i r system can p r e f e r e n t i a l l y
.
.
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e x c i s e a m i s i n c o r p o r a t e d base f r o m t h e t e m p l a t e s t r a n d . W i t h o u t a means t o d i s t i n g u i s h t h e t w o s t r a n d s , t h e nonmutant base o f t h e t e m p l a t e s t r a n d a t a DNA mismatch w o u l d be j u s t as l i k e l y t o be e x c i s e d as i s t h e m u t a n t , m i s i n c o r p o r a t e d base o f t h e n e w l y s y n t h e s i z e d s t r a n d ( r e f s . 68, 69). I n a d d i t i o n t o p a r t i c i p a t i n g i n DNA r e p a i r , dam m e t h y l a t i o n i n E. c o l i has been i m p l i c a t e d i n c o n t r o l l i n g t r a n s c r i p t i o n o f a few genes and i n r e g u l a t i n g t r a n s p o s i t i o n and p o s s i b l y DNA r e p l i c a t i o n ( r e f . 72). Dam-type m e t h y l a t i o n has been f o u n d i n v a r i o u s e n t e r i c b a c t e r i a ( r e f . 73) and m i g h t o c c u r i n some o t h e r t y p e s o f b a c t e r i a a l t h o u g h i t i s c l e a r l y n o t a common phenomenon ( r e f s . 3, 61, 7 4 ) . I t i s i n t e r e s t i n g t o n o t e t h a t t h e s i n g l e - s t r a n d d i s c o n t i n u i t i e s a s s o c i a t e d w i t h t h e n a s c e n t s t r a n d d u r i n g DNA r e p l i c a t i o n appear t o s e r v e t h e mismatch r e p a i r d i r e c t i n g f u n c t i o n o f dam-hemimethyl a t i o n i n Streptococcus pneumoniae, whose DNA i s d e v o i d o f dam m e t h y l a t i o n ( r e f . 7 5 ) . L i ke b a c t e r i a, some 1ower e u k a r y o t e s c o n t a i n modi f ied bases i n t h e i r genomes a1 t h o u g h u n l ike b a c t e r i a no c o r r e s p o n d i n g r e s t r i c t i o n endonucl eases have been r e p o r t e d i n t h e s e o r g a n i s m s . T h e r e f o r e , m o d i f i c a t i o n o f l o w e r e u k a r y o t i c DNAs, 1 i k e t h a t o f v e r t e b r a t e and h i g h e r p l a n t DNAs, p r o b a b l y s e r v e s some m a j o r f u n c t i o n ( s ) o t h e r t h a n e x c l u s i o n o f f o r e i g n DNA o r v i r u s e s . U n i c e l l u l a r e u k a r y o t e s can have m6A as t h e o n l y m i n o r m o d i f i e d b a s e (Tetrahymena thermophila m a c r o n u c l e a r DNA: r e f . 76, 77; O x y t r i c h a f a 1 l o x m a c r o n u c l e a r DNA: r e f . 78; Paramecium a u r e l i a m i c r o n u c l e a r and m a c r o n u c l e a r DNA: r e f . 79), m5C as t h e o n l y (Chlamydomonas m o d i f i e d base ( C h l o r e l l a : r e f . 80), m6A p l u s m5C r h e i n h a r d i : r e f . 81) o r no d e t e c t a b l e DNA m o d i f i c a t i o n ( S a c charomyces c e r e v i s i a e : r e f . 82). I n some d i n o f l a g e l 1a t e s , t h e r e i s a phenomenon n o v e l among e u k a r y o t e s , t h e f o r m a t i o n o f a m o d i f i e d base o t h e r t h a n m5C o r m6A. D i n o f l a g e l 1 a t e DNA has 4-19 m o l % 5 - h y d r o x y m e t h y l u r a c i 1 ( hm5U) depending on t h e s p e c i e s ( r e f . 5). The p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e occurrence i n these u n i c e l l u l a r eukaryotes o f a r a t h e r l a r g e amount o f a DNA base o t h e r w i s e r e s t r i c t e d t o c e r t a i n bact e r i o p h a g e genomes (see above) o r t o f o r m a t i o n as a DNA l e s i o n r e s u l t i n g f r o m r a d i a t i o n damage ( r e f . 83) r e m a i n s t o be d e t e r mined. I n a d d i t i o n t o hm5U, m i n o r amounts o f m5C have been f o u n d
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i n t h e DNA o f two d i n o f l a g e l l a t e species and m6A i n one ( r e f . 84). I n mu1 t i c e l l u l a r eukaryotes, t h e o n l y demonstrated m o d i f i e d base i n t h e DNA i s m5C. I t i s w i d e l y d i s t r i b u t e d among organisms. T h i s base i s p r e s e n t as a m i n o r component i n t h e DNA o f t h e s l i m e mold Physarum polycephalum (1.1 mol% m5C: r e f s . 85, 86) and sea u r c h i n s ( 0.9 mol% m5C; r e f . 87). Although many f u n g i l a c k detect a b l e m o d i f i e d bases i n t h e i r DNA, some such as t h e zygomycete P h y c o m y c e s b 7 a k e s 7 e e a n u s and t h e basidomycete Coprinus cinereus have m5C as a m i n o r base i n t h e i r genome ( r e f . 88-90). Eukaryotes as d i v e r s e as t h e l a t t e r f u n g i , d i n o f l a g e l l a t e s , sea u r c h i n s , s l i m e molds, and v e r t e b r a t e s have t h e i r m5C r e s i d u e s p r e d o m i n a t e l y In some i n CpG d i n u c l e o t i d e s ( r e f s . 84, 91, 92, 88, 93, 1). i n s e c t s , DNA m o d i f i c a t i o n i s u n d e t e c t a b l e . Notably, no m5C has been found by chromatographic a n a l y s i s o f DNA d i g e s t s f r o m D r o s o p h i l a m e l a n o g a s t e r a t t h e l a r v a l o r a d u l t stages ( r e f s . 94, 95). However, m5C i s p r e s e n t i n t h e DNA o f c e r t a i n o t h e r i n s e c t spec i e s . The mealybug c o n t a i n s m5C as t h e o n l y d e t e c t a b l e m o d i f i e d base i n i t s DNA i n amounts dependent upon t h e sex as w e l l as t h e species and l i n e s o f these organisms ( r e f . 96). The observed d i f f e r e n c e s between t h e methyl a t i o n l e v e l s of v a r i o u s mealybug DNAs m i g h t be due t o a r e l a t i o n s h i p between DNA m e t h y l a t i o n and h e t e r o c h r o m a t i n i z a t i o n ( r e f . 96). C e l l s o f these i n s e c t s v a r y i n t h e i r c o n t e n t o f h e t e r o c h r o m a t i c o r supernumerary chromosomes i n a manner c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t i n c r e a s e d DNA methylat i o n i s a s s o c i a t e d w i t h DNA appearing i n t h e h e t e r o c h r o m a t i c f r a c t i o n ( r e f . 96). METHYLATION OF THE DNA OF MITOCHONDRIA, CHLOROPLASTS, AND EUKARYOTIC VIRUSES Higher p l a n t s and v e r t e b r a t e s i n v a r i a b l y c o n t a i n m5C as a m i n o r base i n t h e i r DNA and no o t h e r d e t e c t a b l e m o d i f i e d base ( r e f . 1). I n c o n t r a s t , t h e i r m i t o c h o n d r i a , c h l o r o p l a s t s ( i n t h e case o f p l a n t s ) , and t h e DNA v i r u s e s t h a t i n f e c t t h e s e c e l l s u s u a l l y c o n t a i n l i t t l e o r no DNA m e t h y l a t i o n . For example, polyoma v i r u s ( r e f . 97); adenovirus t y p e 2, t y p e s 5 and 12 ( r e f s . 98101), herpes s i m p l e x v i r u s t y p e 1 ( r e f . 102); herpes s a i m i r i v i r u s ( r e f . 99); and SV40 ( r e f . 101) do n o t c o n t a i n d e t e c t a b l e m o d i f i e d bases d e s p i t e m5C b e i n g p r e s e n t i n t h e i r h o s t DNAs. However, i n 10.4
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tegration of a number of the above DNAs as well as of retroviral provi ral DNA into host genomes in a transcriptional ly repressed form (but not in the transcriptionally active form) is associated with extensive DNA methylation to give m5CpG sites (refs. 104, 105, 106, 107, 108, 109). Although i n f e c t i o u s viral eukaryotic DNAs seem to be generally unmethylated, several exceptions have been reported. DNA from virions of frog virus 3 contains considerable amounts of m5C (ref. 110). Also, viral extrachromosomal DNA from another member of the iridovirus virus family, fish lymphocytosis disease virus, is methylated at 75% of its CpG dinucleotides when isolated from infected tissues (ref. 111). Evidence has been presented for a very slight degree of CpG methyl ation of extrachromosomal human papi 1 loma viral DNA isolated from warts (ref. 112). The extrachromosomal viral DNA in Shope papi 1 loma vi rus-i nfected neoplasms is more highly, but variably, methylated at CpG sites (ref. 113). The only report of a eukaryotic virus associated with a DNA modification other than m5C is a virus called NC-lA, which infects a chlorella-like green alga (ref. 114). This viral DNA contains mA in 5'-GANTC-3' sequences assoc ated with a restriction-modification type system. Its DNA has a high level of m5C (7 mol%) as well as of m6A (7 mol%). Analyses of organelle DNA indicate little or no methylation Chloroplasts from tobacco leaves were reported to contain no detectable m5C in their DNA although the detection limits in this analysis were not indicated (ref. 115). Mitochondria1 DNA from Paramecium a u r e l i a contains (0.1 mol% m5C or m6A; the nuclear DNA also has no detectable m5C but has 2.5 mol% m6A (ref. 79). Less than 0.1 and 0.05 mol% m5C was detectable in the mitochondrial DNA of frog and cultured human (HeLa) cells, respectively (ref. 116). Examination of mitochondria1 DNA from various cultured mammalian cell lines indicated that it contains one-fourth to onefourteenth the m5C content of nuclear DNA (ref. 117). Similarly, we found a low level of m5C (0.14 mol%) in DNA from a mitochondrial fraction of human placenta (ref. 118). Restriction analysis of this DNA indicated (5% contamination with bulk nuclear DNA. However, in these studies of organelle DNA, covalently closed circular DNA is isolated from partially purified subcellular fractions which might be contaminated with small circular DNAs.
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These may c o n t a i n c e r t a i n n u c l e a r DNA sequences t h e r e b y i n t r o ducing contaminating m5C r e s i d u e s . In t h i s regard r e s t r i c t i o n a n a l y s i s of t h e s e o r g a n e l l e DNAs i s l e s s l i a b l e t o f a l s e p o s i t i v e r e s u l t s . We found no d e t e c t a b l e methylation a t 5'-CCGG-3' s i t e s i n human p l a c e n t a l DNA by comparing HpaII and MspI d i g e s t s ( r e f . 119). S i m i l a r l y , no evidence f o r CpG methylation of wheat, y e a s t , Neurospora, r a t , o r c a l f mitochondria1 DNA was found by r e s t r i c t i o n a n a l y s e s ( r e f s . 120, 121). In c o n t r a s t , the u n i c e l l u l a r green a l g a Chlamydomonas methylates almost h a l f o f t h e C r e s i d u e s i n i t s c h l o r o p l a s t DNA i n t h e m a t e r n a l l y derived b u t n o t the p a t e r n a l l y derived c h l o r o p l a s t DNA i n zygotes ( r e f s . 122, 123, 1 2 4 ) . The b i o l o g i c a l impact of t h i s methylation i s u n c l e a r . D I S T R I B U T I O N OF m5C I N THE NUCLEAR DNA OF HIGHER PLANTS AND VERTEBRATES All studied higher p l a n t s c o n t a i n m5C a s a minor b u t cons i d e r a b l e c o n s t i t u e n t of t h e i r n u c l e a r DNA ( 2.3-7.1 mol%) just a s a l l v e r t e b r a t e genomes appear t o have m5C ( 0.7-2.8 mol%) a s t h e i r only minor base ( r e f s . 1, 1 2 5 ) . P l a n t DNAs can have up t o 33% of 10.5
t h e i r C r e s i d u e s methylated (tobacco: r e f . 125) and even higher percentages of the C r e s i d u e s i n c e r t a i n s a t e l l i t e DNAs (melon: r e f . 126; b l u e b e l l : r e f . 127). The d i s t r i b u t i o n o f m5C r e s i d u e s i s sequence-specific ( r e f . 128) with methylation o c c u r r i n g predominately a t CpG o r CpNpG (N i s any base) sequences ( r e f s . 129, 127, 130, 131). Although p l a n t DNA methylation has been s t u d i e d r e l a t i v e l y l i t t l e , there i s a t l e a s t one example of t i s s u e - s p e c i f i c d i f f e r e n c e s i n p l a n t DNA methylation ( r e f . 1 3 2 ) . This observed t i s s u e - s p e c i f i c change i n DNA methylation o f t h e z e i n gene i n d i c a t e s t h a t gene demethylation might, i n some c a s e s , p o s i t i v e l y c o n t r o l gene expression ( r e f . 132). A s t u d y of i n vivo methylation of the transforming plasmid fragment (T-DNA) from Agrobacterium tumefaciens, a b a c t e r i um oncogeni c f o r d i c o t y l donous p l a n t s , s u g g e s t s t h a t methylation of T-DNA i n s i d e p l a n t c e l l s might r e g u l a t e i t s t r a n s c r i p t i o n ( r e f . 133). Another proposed r o l e f o r p l a n t DNA methylation which has experimental s u p p o r t i s t h a t i t might d e c r e a s e t r a n s p o s i t i o n by t r a n s p o s a b l e elements r e s i d e n t i n p l a n t nuclei ( r e f . 134). In c o n s i d e r i n g t h e r o l e s of DNA m e t h y l a t i o n , the mode of f o r -
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mation of m5C residues should be evaluated. Methylation is catalyzed by a cytosine DNA methyl transferase with S-adenosylmethionine and DNA as the substrates after DNA replication (refs. 135-137). The methyl atable ol igonucleotide sequences (CpG and CpNpG) have dyad symmetry, that is, in the 5' ---> 3' direction the same sequence is present on both strands. This facilitates the recognition of plant DNA methylated in only one strand so that such hemimethylated DNA serves as the best DNA substrate for the plant's DNA methyl transferase(s) enzyme(s) (ref. 137). Methylation of hemimethylated DNA is referred to as maintenance methylation because it serves to conserve the pattern of DNA methylation that existed before DNA replication converted a bifilarly (symmetrically) methylated to a hemimethylated site (ref. 1, 127). I n contrast, increases in net DNA methylation that can help establish tissue-specific patterns of DNA methylation or genetic polymorphisms in the population are catalyzed in a de n o w methylation reaction, that is, i n a reaction utilizing a bifilarly unmethylated DNA sequence as the substrate. Similar considerations pertain to vertebrate DNA methylation and vertebrate DNA methyltransferases except that i n this case the site of methylation is predominantly or only CpG (ref. 1). Vertebrate DNAs contain only approximately one-fourth the expected frequency of CpG dinucleotide (ref. 138). Because the majority of CpG sites are methylated and CpG in the predominant site of vertebrate DNA methylation (refs. 2, 104, 139), the limitation on the frequency of mCpG sites is postulated to be due to the accumulation of m5C ---> T transition mutations (refs. 140-144). Both m5C and C residues can deaminate in a heat-induced reaction which should also occur, although to a lower extent, at physiological temperature (refs. 32, 33). Deamination o f an m5C residue yields a T residue which is a normal constituent of DNA unlike the C deamination product, U, which should be efficiently excised from DNA (refs. 32, 145). Furthermore, heat-induced deamination of m5C residues in single-stranded DNA in a physiological buffer occurs at a faster rate than for the analogous C residues (refs. 146, 33). For vertebrates, the extent and pattern of DNA methylation has been shown in many studies to be tissue-specific although, i n
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all cases, CpG dinucleotides appear to be the predominant site of methylation (refs. 1, 147-150, 1 5 2 ) . Generally, the majority of CpG dinucleotides in vertebrate DNA are methylated a1 though the percentage o f unmethylated CpG sites is considerable and varies from tissue to tissue (refs. 1, 150). Not only can the level of DNA methylation differ as much as 30% from one tissue to another and the patterns of DNA methylation vary much within normal cells of an organism, but also, as described below, the amount and distribution of genomic m5C can be highly altered upon tumorigenesis or oncogenic transformation (refs. 153-155). A1 though m5C is generally enriched in heterochromatin-associated DNA in vertebrates, much of it is also in single copy, moderately repeated, and interspersed repeated DNA fractions (refs. 156, 1 5 0 ) . The distribution of unmethylated CpG dinucleotides also shows a tendency toward clustering (refs. 157-159). Some DNA subfractions, like the DNA sequences encoding ribosomal RNA (rDNA) are either unusually m5C-rich (rDNA in fish and amphibi a) or m5C-poor (rDNA i n mammal s , repti 1 es, and bi rds) depending on the type of organism (ref. 141). The methylation status of a DNA sequence can change greatly depending upon whether it is chromosomal or extrachromosomal (refs. 150, 161) or upon its chromosomal location (ref. 139). THE FUNCTIONAL SIGNIFICANCE OF VERTEBRATE DNA METHYLATION: TRANSCRIPTION, CHROMATIN STRUCTURE, DNA REPLICATION AND REPAIR, CANCER AND EMBRYOGENESIS Vertebrate DNA methylation appears to be involved in controlling expression of certain genes. There are many examples of correlations of naturally occurring DNA methyl at ion and the inhibition of gene expression in mammalian and avian genomes (refs. 162-170; and reviewed in ref. 1; 172, 173). In a number of cases it is clear that genetically programmed, decreases in methylation (demethylation) of DNA sequences cannot be sufficient to activate gene expression although they might be necessary for turning on transcription (ref. 174-177). A1 though the mechanisms for methylation-directed transcription control remain to be established, two types of studies have provided insight into this phenomenon. In the first of these, evidence has been presented 10.6
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that a high density of m5C residues affects how DNA is packaged in nucl eosomes and supranucleosome particles (ref. 178, 179). In the second line of experimentation, a DNA-binding protein has been i sol ated from human pl acental nuclei , whi ch speci fi cal ly binds to certain 20-base-pai r DNA sequences only when they are methyl ated at their 2 or 3 CpG sites (refs. 180-183). The high degree of sequence-speci fi ci ty and methyl ati on-dependence of this bi ndi ng reaction suggests that this protein, methylated DNA-binding protein (MDBP), might be a novel transcription regulatory protein specifically responsive to local DNA methylation. Some of the most striking correlations between DNA methylation and silencing of gene expression have been made for mammalian and avian viral genomes or for proviral DNA integrated into mammal i an genomes where they can become methyl ated (refs. 105, 184-192). In some cases, the lack of DNA methylation was shown not to be sufficient for transcriptional activity although evidence suggests that de novo methylation is part of the mechanism for turning off viral genes in the host genome or for keeping them inactive (refs. 193-195). I n v i t r o DNA methylation of a viral promoter (refs. 196, 197) or of the whole proviral DNA (ref. 198) eliminated most or all o f its activity. In one case, methylation caused a shutdown in expression only after a prolonged delay (ref. 199). Demethylation of viral DNAs by 5-azacytidine, which is a pleiotropic antimetabolite that is the strongest known inhibitor of i n v i v o DNA methylation (refs. 200, 201), activates the expression of many viral DNA genomes that have become methylated upon integration into the host genome (refs. 106, 202-205). Similarly, 5-azacytidine or 5-aza-Zt-deoxycytidine treatment turned on expression of many vertebrate genes and induced differentiation in cultured cells (refs. 206-218). Furthermore, d e n o v o methylation of the human 7-globin promoter region prevented its transcription upon introduction into mouse L eel Is, whereas methylation of most of its protein-coding DNA sequence did not (ref. 219). This suggests that methylation at many sites in genes has no effect upon gene expression but at other sites does. This is consistent with our finding that methylation changes during the course of human development at >10 x 106 sites per haploid genome (ref. 150), an extent of change far too great for much of it to be
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invo ved directly in controlling gene expression. Similar results have been found for other mammals (ref. 220). As these results pred ct, a number of tissue-specific differences i n DNA methylation patterns have been found which cannot be correlated with transcriptional activity (refs. 174, 221-223) or which may be consequences rather than causes of transcriptional activation (refs. 224, 225). There are several lines of evidence suggesting that vertebrate DNA methylation plays an important role in determining the structure of chromatin. Satellite DNAs from mammalian tissues, which are found in highly condensed chromatin (heterochromatin), often have a higher m5C content than the low-copy fraction of DNA (refs. 150, 151, 226-230). Heterochromatin is often associated with centromeres. By analysis for immunoreactive sites on metaphase chromosomes from various mammals, m5C residues have been found to be highly enriched in centromeric regions (refs. 231, 232). The distribution of such immunoreactive m5C sites changes during meiosis (ref. 233). Treatment of cell s with 5-azacytidine induces decondensation of heterochromatin (refs. 234-236) as well as the formation of fragile sites and sister-chromatid exchanges (refs. 235-238). In the mealybug, a high mC content in male DNA was correlated with the presence of a paternally derived, genetically inactive facultative heterochromatin (ref. 96). That the methylation status of satellite DNA is physiologically important is suggested by the much higher level of methylation o f several mammalian satellite DNAs in embryoblast (primitive ectoderm-derived) and adult somatic ti ssues than in the extraembryonic trophobl ast and primitive endoderm derivatives or in gametes (refs. 151, 239-241). Not only has DNA methylation been implicated in higher order chromatin structure, but also, m5C residues have been shown to be preferentially associated with nucleosome core, histone H1-associated chromatin (refs. 178, 242). In addition, DNA cytosine methylation can have a large influence on the conformation of the DNA helix, such as, favoring the Z-DNA conformation (ref. 243). These findings suggest that DNA methylation critically influences the condensation of DNA i n chromatin and of certain satellite DNAs in heterochromatin and also may
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subtly affect local chromatin structure. As mentioned above, this, in turn, could be related to the transcriptional activity of DNA and, also, it could affect the ability of chromatin to undergo various types o f recombi nation. DNA methylation has been linked to another form of chromatin condensation namely, the heterochromatinization and transcriptional inactivation of one of the two X chromosome in female mammals. Much evidence suggests that DNA methylation is intimately involved i n this phenomenon (refs. 244-248, 169). Similarly, DNA methylation has been implicated in the late timing of replication of the inactive X chromosome during S phase (refs. 249, 250, 251). It is also possible that DNA methylation plays a role in controlling the initiation of DNA replication as it appears to in Escherichia c o l i (ref. 72). A n additional analogy to bacterial DNA is suggested by evidence that hemimethylation (at C residues) of CpG sites directs mismatch repair in mammalian cells (ref. 252) as does hemimethylation (at A residues of 5'-GATC-3' sites) i n E . c o l i (ref. 75). As described above, there are extensive tissue-specific differences in the amount and distribution of m5C in vertebrate DNA and some of these, probably only a small percentage, are involved i n controlling gene expression. This indicates that DNA methylation probably plays a major role i n guiding differentiation. It is informative, therefore, to compare the methylation status of specific DNA sequences in gametes and somatic cells. A number of vertebrate genes have been shown to be much more methylated at tested CpG sites in sperm DNA than in one or more adult somatic tissue DNAs (refs. 168, 173, 221, 253-255). On the other hand, human sperm DNA has a rather low overall m5C content compared to somatic tissues, which seems to be due to hypomethylation of satellite DNA sequences, as mentioned above, (ref. 220) as well as to hypomethylation of non-satellite (including low-copy-number) sequences which are speci fical ly undermethyl ated in sperm (refs. 158, 159) and probably also i n oocytes (ref. 240). The latter DNA sequences show similar hypermethylation at both 5'-CCGG-3' and 5'-GCGC-3' sites over a long region in all human adult somatic tissues i n contrast to their hypomethyl ation in sperm and intermediate level of methylation in placenta (refs.
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158, 159). The unusual m e t h y l a t i o n p a t t e r n i n t h e s e sequences i n p l a c e n t a i s c o n s i s t e n t w i t h i t s v e r y d i s t i n c t c e l l l i n e a g e and i t s DNA's r a t h e r low o v e r a l l m5C c o n t e n t ( r e f s . 150, 256). The r e s u l t s w i t h sperm DNA suggest t h a t hypomethylation o f many d i s p a r a t e DNA sequences may be necessary f o r gametogenesis o r e a r l y embryogenesis. T h i s may be f o l l o w e d by an i n c r e a s e i n m e t h y l a t i o n o f these sequences e a r l y i n development y i e l d i n g s i m i l a r hypermethylation i n a l l tissues destined t o g i v e r i s e t o the adult. I n a d d i t i o n t o these DNA sequences hypomethylated s p e c i f i c a l l y i n gametes, t h e r e a r e o t h e r a t y p i c a l l y undermethylated DNA sequences which appear t o be undermethylated i n a77 c e l l populat i o n s o f an organism. They a r e sometimes found i n 5 ' gene r e g i o n s n e x t t o a h i g h l y m e t h y l a t e d r e g i o n w i t h i n t h e gene ( r e f . 157). T h i s asymmetri c a l p l acement o f m5CpG s i t e s a1 ong t h e chromosome m i g h t be one s i g n a l f o r i d e n t i f i c a t i o n o f c e r t a i n genes as t r a n scriptional units. A s d e s c r i b e d above, DNA m e t h y l a t i o n i n v e r t e b r a t e s may h e l p i n d i v e r s e ways t o r e g u l a t e macromolecular s y n t h e s i s and d i f f e r e n t i a t i o n . Therefore, derangements i n normal DNA m e t h y l a t i o n c o u l d have p l e i o t r o p i c consequences t o c e l l u l a r p h y s i o l ogy. Indeed, t h e r e i s evidence suggesting t h a t a b n o r m a l i t i e s i n DNA m e t h y l a t i o n a r e i n v o l v e d i n carcinogenesis. Some o f t h i s evidence comes from a n a l y s i s o f d e m e t h y l a t i o n caused b y c a r c i n o g e n i c agents ( r e f s . 165, 205, 257-264). Furthermore, many o f t h e v i r a l DNAs and p r o v i r u s e s whose expression can be c o n t r o l l e d by DNA m e t h y l a t i o n a r e oncogenic v i r u s e s ( r e f s . 106, 107, 185, 187, 196-198, 202, 205). A l s o , s t u d i e s o f human tumors, w i t h o u t t h e p o s s i b l y comp l i c a t i n g intermediacy o f c e l l c u l t u r e , i n d i c a t e a r e l a t i o n s h i p between a1 t e r a t i o n s i n DNA m e t h y l a t i o n and c a r c i n o g e n e s i s . Examination of 23 human c o l o n neoplasms and normal a d j a c e n t i n t e s t i n a l mucosa by Feinberg, V o g e l s t e i n and coworkers ( r e f s . 154, 265, 266) r e v e a l e d t h a t some d i v e r s e genes have a h i g h probabi 1 it y o f becomi ng hypomethyl a t e d d u r i n g f o r m a t i o n o f c o l o n tumors. T h i s was seen i n genes l i k e t h a t o f 7-globin, whose expression ought t o be i r r e l e v a n t t o t h e tumor as w e l l as i n an oncogene, whose m e t h y l a t i o n m i g h t o r m i g h t n o t be o f p h y s i o l o g i c a l s i g n i f i c a n c e i n t h i s tumorigenesis ( r e f s . 154, 266). The hypo-
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methylation of these genes showed much heterogeneity i n its pattern but clearly was not random. In a collaborative study involving C. Gehrke, K. Kuo, A . Feinberg, and one of us (M. Ehrl ich), we have recently demonstrated that the demethylation n these colon tumors, which are known to arise from the essential Y homogeneous cell population of intestinal mucosa, is part of a genome-wide demethylation (ref. 155). Simi 1 arly, chemical ly induced hepatocarcinomas in rats had a lower m5C content than did normal rat liver (ref. 267). Furthe more, despite a wide range of genomic m5C contents, (0.35-1.03% mol% m5C), a statistically significant difference was found in the distribution of DNA methylation levels in various human malignancies, especially in metastases, as compared to that in benign neoplasms or normal human tissues (ref. 153). The percentages of DNA samples with <0.80 mol% m5C or >0.84 mol% m5C were strikingly different for malignancies and for normal tissues or benign tumors (ref. 153). These differences suggest that either hypomethylation often accompanied tumorigenesis or that many of these malignancies were derived from atypical, minor populations of cells with relatively low m5C contents. In the study o f colon tumors mentioned above, we can conclude that decreases in DNA methylation occurred because the tumors are known to arise from the predominant cell type of intestinal mucosa and methylation of tumor DNA was compared to that of the adjacent normal mucosa (ref. 155). The m5C content of neoplastic samples should reflect the percentage of neoplastic cells in the sample, the type of cells which gave rise to the neoplasm, and any changes in DNA methylation which occur during the early stages of oncogenic transformation or during tumor progression. We have proposed that tumor progression with its attendant continually generated cell ul ar diversity is often accompanied by extensive replacement of m5C residues in DNA with cytosine residues (ref, 153). Studies of cultured murine cell s and transplantable tumors support this hypothesis (refs. 260, 261, 268). Hypomethylation of DNA during tumor progression could provide epigenetic changes of the type associated with normal differentiation. It could help establish or mai ntai n transcriptional activity or 1 ead to cancer-re1ated chromosomal rearrangements, gene amplification, and a1 terations in
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chromosome conformation. The resulting changes in expression or replication of the genome might be an important component in the diversification of tumor cells, which allows them to successfully exploit thei r hosts. 10.7 SUMMARY
Most DNAs contain minor amounts of 5-methylcytosine (rn5C), N6-methyladeni ne (m6A), or N4-methyl cytosine (in"). Any one or more of these can be found in most bacterial DNAs. Some lower eukaryotes have m5C, m6A or both as a minor base in their genomes. In the DNA of all studied vertebrates and higher plants, m5C, and only m5C, is the naturally occurring modified base. The levels and patterns of m5C in vertebrate genomes are ti ssue-speci fi c. Some, but clearly not most, vertebrate DNA methylation has been linked to the regulation of gene transcription. As for m6A in the Escherichia c o l i genome, m5C in the DNA of eukaryotes may function in controlling DNA repair, replication, and rearrangements as well as in control of transcription. However, although bacterial DNA methylation is often involved in r e s t r i c t i o n - m o d i f i c a t i o n systems, there is no evidence for this function in the case of vertebrate DNA methylation. Vertebrate DNA methylation may, however, be an important determinant of chromatin structure. In addition to methylated bases present as minor DNA components, other modified bases are major components of certain bacteriophage genomes. These have one of their bases, usually a pyrimidine, largely or completely replaced by the modified derivative. The derivatives include gl ucosyl ated 5-hydroxy-methylcytosine, uracil, 5-hydroxymethyluracil, 5-(4-aminobutylaminomethy1)uraci 1 , and gl ucosyl ated and gl ucuroni c aci d-l-phosphorylated 5-(4',5'-dihydroxypenty1)uracil. These major modifications of phage genomes appear to help, sometimes in subtle ways, the virus to distinguish its genome from that of the host in its exploitation of the host cell and to evade host DNA restriction systems . 10.8
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ts
CHAPTER 10 NATURALLY OCCURRING MODIFIED NUCLEOSIDES I N DNA MELANIE EHRLICH and XIAN-YANG ZHANG Department o f Biochemistry, Tulane Nedical School, New Orleans, LA 70112, U . S . A .
TABLE 10.1 10.2 10.3
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OF CONTENTS Introduction H i g h l y M o d i f i e d Bacteriophage DNA M o d i f i e d Bases i n DNA f r o m B a c t e r i a and Lower Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . Meth l a t i o n o f t h e DNA o f M i t o c h o n d r i a , C h l o r o p l a s t s , and h k a r y o t i c V i r u s e s D i s t r i b u t i o n o f m5C i n t h e N u c l e a r DNA o f H i g h e r P l a n t s and V e r t e b r a t e s The F u n c t i o n a l S i g n i f i c a n c e o f V e r t e b r a t e DNA Meth 1a t i o n : T r a n s c r i p t i o n , Chromatin S t r u c t u r e , DNA R e p r i c a t i o n and Repair, Cancer and Embryogenesis Summary References
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INTRODUCTION To s t a t e t h a t DNA i s composed o f o n l y f o u r bases, adenine (A) t thymine (T), guanine (G) and c y t o s i n e (C) i s an o v e r - s i m p l i f i c a t i o n because most t y p e s o f DNA n a t u r a l l y c o n t a i n one t o t h r e e a d d i t i o n a l bases, m o d i f i e d forms o f A o r C. U s u a l l y , o n l y a m i n o r The p o r t i o n o f t h e C o r A r e s i d u e s i s m o d i f i e d ( r e f s . 1-3). e x c e p t i o n s a r e t h e DNA from c e r t a i n b a c t e r i o p h a g e s ( r e f . 4) and from some d i n o f l a g e l l a t e s ( r e f . 5 ) . One o r a n o t h e r o f a v a r i e t y o f m o d i f i e d p y r i m i d i n e s a r e found i n t h e s e phage DNAs and 5h y d r o x y m e t h y l u r a c i l i n d i n o f l a g e l l a t e DNA. I n c o n t r a s t , t h e o n l y m o d i f i e d bases found i n a1 1 o t h e r DNAs a r e 5-methyl cytosine (rn5C), N4-methylcytosine (m4C), and N6-methyladenine (m6A). The f u n c t i o n s o f t h e s e DNA m o d i f i c a t i o n s a r e d i v e r s e and range f r o m p r o t e c t i o n o f b a c t e r i a l h o s t DNA a g a i n s t a pathway f o r d e g r a d i n g f o r e i g n DNA t o c o n t r o l o f p r o k a r y o t i c and e u k a r y o t i c t r a n s c r i p t i o n a l a c t i v i t y .
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HIGHLY MODIFIED BACTERIOPHAGE DNA Some t y p e s of b a c t e r i a l viruses, although not most, have one of the DNA bases completely o r l a r g e l y replaced by a modified derivative (ref. 4). These highly modified phage DNAs almost always c o n t a i n pyrimidine m o d i f i c a t i o n s . The f i r s t of these t o be di scovered was 5-hydroxymethyl c y t o s i ne (hm5C) i n phage T2, T4, and T6 DNAs ( r e f . 6 ) . These e n t e r i c Escherichia c o y i phages, which were among the e a r l i e s t s u b j e c t s of i n t e n s i v e molecular b i o l o g i c a l i n v e s t i g a t i o n , a r e very highly modified, a f a c t which sometimes complicated t h e i r use a s experimental models. Not only do they have a hydroxymethyl group a t each C r e s i d u e , b u t a l s o , 75-100% o f t h e i r r e s u l t i ng hm5C r e s i d u e s a r e gl ucosyl a t e d ( r e f s . 7-9). The type of l i n k a g e of t h e glucose and t h e number of g l u c o s e m o i e t i e s per hm5C r e s i d u e depends upon t h e phage type ( r e f . 1 0 ) . In T2 phage DNA, 70% of t h e hm5C r e s i d u e s c o n t a i n an a-glucosyl moiety, 5% have a g e n t i o b i o s e (8-91 ucosyl -a-gl ucosyl) , and the rest a r e nonglucosylated. Phage T4 and T6 DNAs have the f o l l o w i n g d i s t r i b u t i o n s of t h e i r hm5C r e s i d u e s : T4, 70% a - g l u c o s y l a t e d , 30%-8glucosyl a t e d , and 0% nongl ucosyl a t e d ; T6, 3% 0-91 ucosyl a t e d , 72% 8-1,6-91 ucosyl -a-gl ucosyl a t e d ( g e n t i o b i o s e ) , and 25% nongl ucosyla t e d . The formation of t h e hydroxymethyl group i s c a t a l y z e d a t t h e deoxymononucl e o t i d e 1 eve1 by a phage-encoded dCMP hydroxymethylt r a n s f e r a s e p r i o r t o i n c o r p o r a t i o n i n t o the DNA ( r e f . 1 1 ) . The s y n t h e s i s of this phage DNA e x p l o i t s t h e h o s t ' s r e s o u r c e s by degrading h o s t DNA and dCTP and converting t h e r e s u l t i n g recycled dCMP a s well a s de nova synthesized dCMP t o hm5dCTP. This phosphorylation involves a phage-encoded deoxynucleoside monophosphate kinase and a host diphosphate kinase a s well a s the phage hydroxymethyl t r a n s f e r a s e ( r e f . 1 1 ) . A f t e r i n c o r p o r a t i o n of the hm5dCMP r e s i d u e s , the gl ucosyl a t i o n of t h e hydroxymethyl groups i nvol ves hmT-speci f i c a- and 8-gl ucosyl t r a n s f e r a s e s , which have some sequence s p e c i f i c i t y ( r e f . 1 2 ) . This g l u c o s y l a t i o n i s normally necessary t o prevent t h e degradation of these phage DNAs by E . c o l i nucleases a s well a s by phage-encoded n u c l e a s e s meant t o d e s t r o y h o s t DNA ( r e f s . 13-16). I f dCMP r e s i d u e s a r e i n c o r p o r a t e d i n t o phage DNA, the DNA w i l l be degraded by the T4-encoded endonuclease I 1 ( r e f . 1 6 ) . Therefore, hydroxymethyl a t i o n and glucos y l a t i o n of c y t o s i n e r e s i d u e s i n T-even phage p l a y an important 10.2
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r o l e i n t h e phage l i f e c y c l e by p r o t e c t i n g phage DNA f r o m degradat i o n w h i l e a l l o w i n g h o s t DNA t o be s p e c i f i c a l l y r e c o g n i z e d f o r such h y d r o l y s i s . I t a l s o f a c i l i t a t e s p h a g e - s p e c i f i c t r a n s c r i p t i o n l a t e i n i n f e c t i o n c a t a l y z e d by a phage-modified h o s t RNA p o l y merase ( r e f s . 4, 17, 18). L i k e many o t h e r types o f b a c t e r i o p h a g e DNAs i n c l u d i n g t h o s e which a r e n o t h i g h l y m o d i f i e d ( r e f s . 19, 20), T4 DNA c o n t a i n s m i n o r amounts o f m6A ( r e f . 21). T h i s m e t h y l a t i o n o f A r e s i d u e s i s c a t a l y z e d by a T4-induced, sequence-specific DNA methyl t r a n s f e r a s e ( r e f . 21). The p h y s i o l o g i c a l s i g n i f i c a n c e o f m i n o r amounts o f m6A o r m5C i n phage DNAs remains t o be d e t e r mined. E x t e n s i v e hydroxymethyl a t i o n occurs a t a n o t h e r p y r i m i d i n e r e s i d u e i n a s e t o f c l o s e l y r e l a t e d phage DNAs which a r e widespread i n s o i l ( r e f . 22). A few p a r t i a l l y homologous phages t h a t i n f e c t Bacillus subtilis, SPO1, ue, SP8, SP82G, 2C, and SP5, have t h e T r e s i d u e s i n t h e i r genomes c o m p l e t e l y r e p l a c e d by 5-hydroxym e t h y l u r a c i l (hm5U) r e s i d u e s ( r e f s . 11, 22) which a r e nonglucosy1 ated. L i k e t h e T-even phages, t h e s e phages hydroxymethylate a t t h e mononucleotide l e v e l , i n t h i s case, w i t h dUMP as t h e s u b s t r a t e . The r e a c t i o n i s s i m i l a r l y c a t a l y z e d by a phage-induced hydroxymethylase ( r e f . 23). To p r e v e n t i n c o r p o r a t i o n of dTTP, t h e phages encode a dTTPase ( r e f . 24) as w e l l as an i n h i b i t o r o f t h e h o s t b a c t e r i u m ' s t h y m i d y l a t e s y n t h e t a s e ( r e f . 2 3 ) . Channeling of p y r i m i d i n e s t o s y n t h e s i s o f hm5dUTP i s f a c i l i t a t e d by two phageinduced enzymes, a dCMP deaminase and a deoxynucleoside monophosphate k i n a s e ( r e f s . 4, 23). I n h i b i t i o n o f h o s t DNA r e p l i c a t i o n occurs a f t e r i n f e c t i o n i n i t i a l l y as t h e r e s u l t o f a phage-induced i n h i b i t o r o t h e r than t h e i n h i b i t o r o f t h y m i d y l a t e s y n t h e t a s e ( r e f . 22). U n l i k e t h e case f o r t h e T-even phages, h o s t DNA i s n o t e x t e n s i v e l y degraded and t h e hm5U-containing B . s u b t i l i s phages can have as much as 20% o f t h e i r hm5U r e p l a c e d by T w i t h o u t l o s i n g t h e i r v i a b i l i t y ( r e f . 25). Nonetheless, t r a n s c r i p t i o n i n t h e m i d d l e and l a t e stages o f t h e i n f e c t i o u s c y c l e r e q u i r e s hm*Uc o n t a i n i n g phage DNA sequences as w e l l as a phage-encoded regul a t o r y p r o t e i n r e p l a c i n g one of t h e h o s t RNA polymerase's sigma f a c t o r s ( r e f . 26). I n a d d i t i o n , a DNA-binding p r o t e i n which b i n d s s p e c i f i c a l l y t o c e r t a i n hm5U-containing sequences i s s y n t h e s i z e d ( r e f . 27).
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Another t y p e o f B. subtilis phage has a d i f f e r e n t s u b s t i t u e n t r e p l a c i n g t h e T r e s i d u e s of i t s DNA, namely u r a c i l (U) r e s i d u e s . J u s t as T r e s i d u e s a r e commonly found i n RNA ( t R N A ) a l t h o u g h t h e y a r e m o s t l y a s s o c i a t e d w i t h DNA, so U r e s i d u e s can be a normal c o n s t i t u e n t o f DNA. To date, g e n e t i c a l l y programmed f o r m a t i o n o f U r e s i d u e s i n DNA has been found o n l y i n phage PBSl and i t s v e r y c l o s e l y r e l a t e d d e r i v a t i v e PBS2 ( r e f s . 28, 29). PBS2 has been shown t o encode a dTMP 5'-phosphatase, which h e l p s p r e v e n t T r e s i d u e s from b e i n g i n c o r p o r a t e d i n t o phage DNA ( r e f . 30), and a dUMP kinase, which t o g e t h e r w i t h a h o s t n u c l e o s i d e diphosphate k i n a s e p r o v i d e s dUTP f o r phage DNA s y n t h e s i s . The phage d u r i n g i t s l i f e c y c l e has t o cope w i t h h o s t mechanisms f o r e x c l u d i n g advent i t i o u s U r e s i d u e s from DNA, namely, a h o s t dUTPase and a h o s t uracil-DNA g l y c o s y l a s e ( r e f . 31). The l a t t e r enzyme e x c i s e s t h e n o r m a l l y small amount o f m i s i n c o r p o r a t e d dUTP (dUMP r e s i d u e s ) as w e l l as U r e s i d u e s a r i s i n g from spontaneously o c c u r r i n g heatinduced deamination o f C r e s i d u e s ( r e f s . 32, 33). To c o u n t e r a c t t h e a c t i v i t y o f t h e s e enzymes, phage i n f e c t i o n induces t h e synt h e s i s o f i n h i b i t o r s o f t h e DNA-uracil e x c i s i o n pathway and o f dUTPase ( r e f s . 31, 34, 35). Another phage t h a t i n f e c t s B. subtilis has a c o m p l i c a t e d and unique t y p e o f m o d i f i c a t i o n i n i t s DNA. Phage SP15 DNA c o n t a i n s 60% o f i t s T r e s i d u e s r e p l a c e d by 5 - ( 4 ' ,5'-dihydroxypenty1)uracil (hpsU) ( r e f . 36) i n which a f i v e - c a r b o n s i d e c h a i n w i t h two hydroxyl groups i s bonded t o t h e 5 p o s i t i o n o f t h e p y r i m i d i n e , t h e same p o s i t i o n m o d i f i e d i n a l l t h e above-mentioned p y r i m i d i n e s . Attached t o one o f t h e h y d r o x y l groups i s a glucose m o e i t y and t o t h e o t h e r v i a a phosphodiester l i n k a g e i s a phosphoglucuronate ( r e f . 37). T h i s g l u c u r o n i c acid-l-phosphate m o i e t y i n t r o d u c e s a phosphate t h a t i s n o t p a r t o f t h e DNA's phosphodiester backbone. I t c o n f e r s e x t r a n e g a t i v e changes on t h e DNA and i s a p p a r e n t l y responsi b l e f o r t h e a1 k a l ine 1a b i 1i t y and 1ow me1ti ng temperature o f t h i s DNA ( r e f . 38). SP15 DNA i s t h e o n l y known DNA s t a b l y c o n t a i n i n g phosphates t h a t a r e n o t p a r t o f t h e phosphodiester backbone. The d i s t r i b u t i o n o f hp5U versus T r e s i d u e s i n SP15 DNA i s .nonrandom. How hp5U, which o n l y partially r e p l a c e s T i n t h i s DNA, i s i n t r o d u c e d i n t o s p e c i f i c p o s i t i o n s remains unknown ( r e f . 37). Less w e l l d e f i n e d c h e m i c a l l y i s t h e h y p e r m o d i f i e d T d e r i v a -
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t i v e which p a r t i a l l y r e p l a c e s T i n t h e B. subtilis phage S P l O DNA. The s y n t h e s i s o f t h i s DNA i n v o l v e s a novel pathway i n which hm5U r e s i d u e s ( f r o m hm5dUTP) a r e i n c o r p o r a t e d i n t o t h e DNA and t h e n is pyrophosphorylated. Subsequently, 15% of i t l o s e s t h e pyrophosphate and i s converted t o t h e hypermodified T r e s i d u e s and t h e r e s t i s converted t o T r e s i d u e s ( r e f . 39). A s i m i l a r pathway i s encoded by phage dW14 t h a t i n f e c t s Pseudomonas acidovorans. The DNA o f t h i s phage has h a l f o f i t s T r e s i d u e s r e p l aced by 5- (4-ami n o b u t y l ami nomethyl ) u r a c i 1 (a-putresc i n y l t h y m i n e ; putT) which c o n f e r s a p o s i t i v e charge on t h e r e s i d u e ( r e f . 4 ) . T h i s base i s r e s p o n s i b l e f o r t h e higher-than-expected m e l t i n g temperature o f t h e DNA and i t s a l k a l i l a b i l i t y ( r e f . 40). Both putT and T i n dW14 DNA a r i s e from hm5dUTP. A f t e r dW14 i n f e c t i o n , t h e h o s t s u f f e r s t h e gradual l o s s o f dTTP and t h e appearance o f hm5dUTP c a t a l y z e d by a phage-induced enzyme ( r e f . 41). T h i s hm5dUTP i s i n c o r p o r a t e d i n t o t h e DNA and, a t t h e p o l y n u c l e o t i d e l e v e l , pyrophosphorylated ( r e f . 42). As f o r t h e T r e s i d u e s o f SPlO, T r e s i d u e s i n dW14 a r e formed a t t h e DNA l e v e l f r o m 5[(hydroxymethyl)-0-pyrophosphoryl] u r a c i 1 r e s i d u e s ( r e f s . 41, 42). The d i s t r i b u t i o n o f putT, which o n l y p a r t i a l l y r e p l a c e s i t s corresponding unmodified m a j o r base, i s s e q u e n c e - s p e c i f i c ( r e f . 43) as i n t h e case of hp5U i n SP15 DNA. Two more examples o f unusual bases i n h i g h l y m o d i f i e d phage DNAs have been r e p o r t e d . One i s a C d e r i v a t i v e , 2,5-dihydroxy-4ami nopyrimi d i n e , (5-hydroxycytosi ne) , whi ch c o m p l e t e l y r e p l aces C i n t h e DNA o f phage N-17, whose h o s t i s Shigella flexneri ( r e f . 44). The o t h e r i s t h e o n l y r e p o r t e d example o f a h i g h l y m o d i f i e d DNA c o n t a i n i n g as a m a j o r base a p u r i n e d e r i v a t i v e . T h i s i s found i n cyanophage S-2L, which i n f e c t s t h e b l ue-green a l g a e S y n e c h o c o c c u s e l o n g a t u s . I t has been i d e n t i f i e d as 2-aminoadenine and c o m p l e t e l y r e p l a c e s A ( r e f . 45). The o n l y o t h e r example o f extens i v e , b u t n o t major, replacement o f a p u r i n e i n DNA i s found i n E . c o l i phage Mu DNA. T h i s phage encodes t h e c o n v e r s i o n o f 15% o f i t s A r e s i d u e s t o a-N-(9-~-0-2'-deoxyri b o f u r a n o s y l p u r i n-6-y1)glycinamide i n which an acetamido group i s on t h e N-6 o f adenine ( r e f . 46). A l l o f t h e above-mentioned phage DNAs have as t h e i r main m o d i f i e d base a d e r i v a t i v e n o t commonly found i n o t h e r DNAs. Only
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one o f t h e s e , hm5U, has been f o u n d i n any DNA o t h e r t h a n t h a t o f phage (see b e l o w ) . There i s one example o f a h i g h l y m o d i f i e d DNA, phage XP12 DNA, whose m o d i f i e d base i s a m a j o r component o f t h i s phage DNA and a m i n o r component o f many o t h e r DNAs. Phage XP12 was d i s c o v e r e d b y Tsong-Teh Kuo g r o w i n g on t h e p l a n t p a t h o g e n X a n t h o m o n a s oryzae ( r e f . 47). XP12 DNA has e s s e n t i a l l y a l l o f i t s C r e s i d u e s r e p 1 aced b y 5-methyl c y t o s i n e (m5C) ( r e f . 48-50). T h i s base i s a l s o f o u n d as a m i n o r component o f v e r y many t y p e s o f DNAs (see b e l o w ) . The m5C r e s i d u e s g i v e XP12 DNA a m e l t i n g t e m p e r a t u r e h i g h e r t h a n expected; t h e r e f o r e , t h e 5-methyl a t i o n i n c r e a s e s t h e s t a b i l i t y o f t h e h e l i x ( r e f . 49). The s o u r c e o f t h e m e t h y l g r o u p f o r XP12 D N A ' s m5C r e s i d u e s (and f o r T r e s i d u e s , i n g e n e r a l ) i s t h e 3-carbon o f s e r i n e r a t h e r t h a n t h e t h i o m e t h y l c a r b o n o f methi o n i n e as i n t h e f o r m a t i o n o f m5C as a m i n o r base i n DNA ( r e f . 49). M e t h y l a t i o n o f t h e C r e s i d u e s f o r XP12 DNA o c c u r s a t t h e m o n o n u c l e o t i d e l e v e l i n a r e a c t i o n c a t a l y z e d b y a phage-induced d e o x y c y t i d y l a t e m e t h y l t r a n s f e r a s e ( r e f . 51). XP12 i n f e c t i o n i n d u c e s t h e s y n t h e s i s o f a 5-methyldeoxyc y t i d i n e 5 ' - monophosphate k i n a s e , which, u n l i k e E . c o l i and X . oryzae m o n o n u c l e o t i d e k i n a s e s , can c a t a l y z e t h e p h o s p h o r y l a t i o n o f m5dCMP ( r e f . 52). The c o n v e r s i o n o f m5dCDP t o m5dCTP i s app a r e n t l y catalyzed by a r e l a t i v e l y n o n s p e c i f i c h o s t nucleoside d i p h o s p h a t e k i n a s e ( r e f . 52). I n t e r e s t i n g l y , XP12 u t i l i z e s t h e m5dCTP i n a u n i q u e pathway f o r p r o d u c t i o n o f T r e s i d u e s . I t i n d u c e s t h e f o r m a t i o n o f an mSdCTP deaminase t h a t g e n e r a t e s dTTP b y d e a m i n a t i o n o f p a r t o f t h e p o o l o f n e w l y c r e a t e d m5dCTP i n p h a g e - i n f e c t e d c e l l s ( r e f . 53). The phage a l s o i n d u c e s t h e synt h e s i s o f an e x o n u c l e a s e w i t h a p r e f e r e n c e f o r d o u b l e - s t r a n d e d DNA ( r e f . 5 4 ) . However, t h i s enzyme h y d r o l y z e s XP12 DNA and h o s t DNA e q u a l l y w e l l . T h i s c o n t r a s t s w i t h a h o s t e x o n u c l e a s e t h a t has a p r e f e r e n c e f o r s i n g l e - s t r a n d e d DNA and h y d r o l y z e s h o s t DNA much b e t t e r t h a n XP12 DNA ( r e f . 54, 55). Thus, some phage-induced enzymes i n v o l v e d i n m e t a b o l i s m o f h i g h l y m o d i f i e d phage DNAs may n o t have a s e l e c t i v i t y f o r t h e m o d i f i e d DNA, b u t r a t h e r may j u s t n o t be i n h i b i t e d by t h e m o d i f i c a t i o n . 10.3
MODIFIED BASES I N DNA FROM BACTERIA AND LOWER EUKARYOTES B a c t e r i a u s u a l l y c o n t a i n one o r more o f t h e f o l l o w i n g m e t h y l -
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ated bases i n t h e i r DNA: m6A, m5C, and t h e newly d i s c o v e r e d m4C ( r e f s . 3, 56-61). These m e t h y l a t e d bases u s u a l l y c o n s t i t u t e 0.010.7% o f t h e r e s i d u e s of t h e DNA, a l t h o u g h 2 mol% m6A i s p r e s e n t i n one b a c t e r i a l genome ( r e f s . 3, 61, 62). The r e l a t i v e frequency o f occurrence o f m i n o r amounts these m o d i f i e d bases i n b a c t e r i a l DNAs i s m6A > m5C > m4C ( r e f . 61). Also, i t i s o n l y m6A t h a t has been found a t l e v e l s > 0 . 4 mol% ( r e f s . 3, 61). These a r e t h e o n l y observed m o d i f i e d bases i n b a c t e r i a l DNA and a l t h o u g h t h e g r e a t m a j o r i t y o f s t u d i e d b a c t e r i a c o n t a i n a t l e a s t one o f them i n t h e i r genomes, some b a c t e r i a l DNAs have no d e t e c t a b l e m o d i f i e d bases ( r e f s . 3, 61). Much o f t h i s b a c t e r i a l DNA m e t h y l a t i o n i s p a r t o f r e s t r i c t i o n / m o d i f i c a t i o n systems ( r e f . 63). These i n v o l v e sequence-speci f i c adenine- o r c y t o s i ne-DNA methyl t r a n s f e r a s e s and corresponding sequence-speci f i c endonucl eases ( r e s t r i c t i o n endonucleases) R e s t r i c t i o n endonucleases general l y c l e a v e t h e same o l i g o n u c l e o t i d e recognized by t h e methyl t r a n s f e r a s e and w i 11 c l e a v e t h e sequence o n l y i n i t s unmethylated form. However, i n a t l e a s t one case, a r e s t r i c t i o n endonuclease c l e a v e s o n l y t h e a p p r o p r i a t e l y methylated r e c o g n i t i o n sequences r a t h e r than t h e unmethylated sequence ( r e f . 64). The restriction/modification systems s e r v e t o demarcate h o s t DNA as s e l f and a l l o w phage DNA which had i n f e c t e d a b a c t e r i a l s t r a i n w i t h a h e t e r o l o g o u s m o d i f i c a t i o n system o r o t h e r f o r e i g n DNA e n t e r i n g t h e b a c t e r i a l c e l l t o be recognized as n o n - s e l f and thereby, be degraded ( r e f s . 65, 66). However, t h e f u n c t i o n s o f t h e m o d i f i c a t i o n and r e s t r i c t i o n enzymes may not be l i m i t e d t o e x c l u s i o n o f f o r e i g n DNA. Also, some bact e r i a1 DNA methyl t r a n s f e r a s e s recogni ze s p e c i f i c DNA sequences f o r which no corresponding r e s t r i c t i o n enzyme e x i s t s i n t h e h o s t bacterium. The most w e l l s t u d i e d example o f t h e s e i s t h e DNA adenine m e t h y l a t i o n (dam) system o f E. coli which c o n v e r t s 5 ' GATC-3 ' sequences t o 5 ' -Gm6ATC-3 ' ( r e f . 67) Dam m e t h y l a t i o n d i r e c t s DNA mismatch r e p a i r as f o l l o w s ( r e f s . 68, 69). The r e c o g n i t i o n sequence i s a palindrome so t h a t n o r m a l l y b o t h s t r a n d s o f t h e DNA a r e s y m m e t r i c a l l y m e t h y l a t e d a t 5'-GATC-3' s i t e s . However, immediately a f t e r DNA s y n t h e s i s o n l y one s t r a n d , t h e t e m p l a t e s t r a n d , i s m e t h y l a t e d ( r e f s . 70, 71). T h i s hemim e t h y l a t i o n a l l o w s t h e nascent s t r a n d t o be d i s t i n g u i s h e d from t h e t e m p l a t e so t h a t t h e DNA mismatch r e p a i r system can p r e f e r e n t i a l l y
.
.
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e x c i s e a m i s i n c o r p o r a t e d base f r o m t h e t e m p l a t e s t r a n d . W i t h o u t a means t o d i s t i n g u i s h t h e t w o s t r a n d s , t h e nonmutant base o f t h e t e m p l a t e s t r a n d a t a DNA mismatch w o u l d be j u s t as l i k e l y t o be e x c i s e d as i s t h e m u t a n t , m i s i n c o r p o r a t e d base o f t h e n e w l y s y n t h e s i z e d s t r a n d ( r e f s . 68, 69). I n a d d i t i o n t o p a r t i c i p a t i n g i n DNA r e p a i r , dam m e t h y l a t i o n i n E. c o l i has been i m p l i c a t e d i n c o n t r o l l i n g t r a n s c r i p t i o n o f a few genes and i n r e g u l a t i n g t r a n s p o s i t i o n and p o s s i b l y DNA r e p l i c a t i o n ( r e f . 72). Dam-type m e t h y l a t i o n has been f o u n d i n v a r i o u s e n t e r i c b a c t e r i a ( r e f . 73) and m i g h t o c c u r i n some o t h e r t y p e s o f b a c t e r i a a l t h o u g h i t i s c l e a r l y n o t a common phenomenon ( r e f s . 3, 61, 7 4 ) . I t i s i n t e r e s t i n g t o n o t e t h a t t h e s i n g l e - s t r a n d d i s c o n t i n u i t i e s a s s o c i a t e d w i t h t h e n a s c e n t s t r a n d d u r i n g DNA r e p l i c a t i o n appear t o s e r v e t h e mismatch r e p a i r d i r e c t i n g f u n c t i o n o f dam-hemimethyl a t i o n i n Streptococcus pneumoniae, whose DNA i s d e v o i d o f dam m e t h y l a t i o n ( r e f . 7 5 ) . L i ke b a c t e r i a, some 1ower e u k a r y o t e s c o n t a i n modi f ied bases i n t h e i r genomes a1 t h o u g h u n l ike b a c t e r i a no c o r r e s p o n d i n g r e s t r i c t i o n endonucl eases have been r e p o r t e d i n t h e s e o r g a n i s m s . T h e r e f o r e , m o d i f i c a t i o n o f l o w e r e u k a r y o t i c DNAs, 1 i k e t h a t o f v e r t e b r a t e and h i g h e r p l a n t DNAs, p r o b a b l y s e r v e s some m a j o r f u n c t i o n ( s ) o t h e r t h a n e x c l u s i o n o f f o r e i g n DNA o r v i r u s e s . U n i c e l l u l a r e u k a r y o t e s can have m6A as t h e o n l y m i n o r m o d i f i e d b a s e (Tetrahymena thermophila m a c r o n u c l e a r DNA: r e f . 76, 77; O x y t r i c h a f a 1 l o x m a c r o n u c l e a r DNA: r e f . 78; Paramecium a u r e l i a m i c r o n u c l e a r and m a c r o n u c l e a r DNA: r e f . 79), m5C as t h e o n l y (Chlamydomonas m o d i f i e d base ( C h l o r e l l a : r e f . 80), m6A p l u s m5C r h e i n h a r d i : r e f . 81) o r no d e t e c t a b l e DNA m o d i f i c a t i o n ( S a c charomyces c e r e v i s i a e : r e f . 82). I n some d i n o f l a g e l 1a t e s , t h e r e i s a phenomenon n o v e l among e u k a r y o t e s , t h e f o r m a t i o n o f a m o d i f i e d base o t h e r t h a n m5C o r m6A. D i n o f l a g e l 1 a t e DNA has 4-19 m o l % 5 - h y d r o x y m e t h y l u r a c i 1 ( hm5U) depending on t h e s p e c i e s ( r e f . 5). The p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e occurrence i n these u n i c e l l u l a r eukaryotes o f a r a t h e r l a r g e amount o f a DNA base o t h e r w i s e r e s t r i c t e d t o c e r t a i n bact e r i o p h a g e genomes (see above) o r t o f o r m a t i o n as a DNA l e s i o n r e s u l t i n g f r o m r a d i a t i o n damage ( r e f . 83) r e m a i n s t o be d e t e r mined. I n a d d i t i o n t o hm5U, m i n o r amounts o f m5C have been f o u n d
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i n t h e DNA o f two d i n o f l a g e l l a t e species and m6A i n one ( r e f . 84). I n mu1 t i c e l l u l a r eukaryotes, t h e o n l y demonstrated m o d i f i e d base i n t h e DNA i s m5C. I t i s w i d e l y d i s t r i b u t e d among organisms. T h i s base i s p r e s e n t as a m i n o r component i n t h e DNA o f t h e s l i m e mold Physarum polycephalum (1.1 mol% m5C: r e f s . 85, 86) and sea u r c h i n s ( 0.9 mol% m5C; r e f . 87). Although many f u n g i l a c k detect a b l e m o d i f i e d bases i n t h e i r DNA, some such as t h e zygomycete P h y c o m y c e s b 7 a k e s 7 e e a n u s and t h e basidomycete Coprinus cinereus have m5C as a m i n o r base i n t h e i r genome ( r e f . 88-90). Eukaryotes as d i v e r s e as t h e l a t t e r f u n g i , d i n o f l a g e l l a t e s , sea u r c h i n s , s l i m e molds, and v e r t e b r a t e s have t h e i r m5C r e s i d u e s p r e d o m i n a t e l y In some i n CpG d i n u c l e o t i d e s ( r e f s . 84, 91, 92, 88, 93, 1). i n s e c t s , DNA m o d i f i c a t i o n i s u n d e t e c t a b l e . Notably, no m5C has been found by chromatographic a n a l y s i s o f DNA d i g e s t s f r o m D r o s o p h i l a m e l a n o g a s t e r a t t h e l a r v a l o r a d u l t stages ( r e f s . 94, 95). However, m5C i s p r e s e n t i n t h e DNA o f c e r t a i n o t h e r i n s e c t spec i e s . The mealybug c o n t a i n s m5C as t h e o n l y d e t e c t a b l e m o d i f i e d base i n i t s DNA i n amounts dependent upon t h e sex as w e l l as t h e species and l i n e s o f these organisms ( r e f . 96). The observed d i f f e r e n c e s between t h e methyl a t i o n l e v e l s of v a r i o u s mealybug DNAs m i g h t be due t o a r e l a t i o n s h i p between DNA m e t h y l a t i o n and h e t e r o c h r o m a t i n i z a t i o n ( r e f . 96). C e l l s o f these i n s e c t s v a r y i n t h e i r c o n t e n t o f h e t e r o c h r o m a t i c o r supernumerary chromosomes i n a manner c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t i n c r e a s e d DNA methylat i o n i s a s s o c i a t e d w i t h DNA appearing i n t h e h e t e r o c h r o m a t i c f r a c t i o n ( r e f . 96). METHYLATION OF THE DNA OF MITOCHONDRIA, CHLOROPLASTS, AND EUKARYOTIC VIRUSES Higher p l a n t s and v e r t e b r a t e s i n v a r i a b l y c o n t a i n m5C as a m i n o r base i n t h e i r DNA and no o t h e r d e t e c t a b l e m o d i f i e d base ( r e f . 1). I n c o n t r a s t , t h e i r m i t o c h o n d r i a , c h l o r o p l a s t s ( i n t h e case o f p l a n t s ) , and t h e DNA v i r u s e s t h a t i n f e c t t h e s e c e l l s u s u a l l y c o n t a i n l i t t l e o r no DNA m e t h y l a t i o n . For example, polyoma v i r u s ( r e f . 97); adenovirus t y p e 2, t y p e s 5 and 12 ( r e f s . 98101), herpes s i m p l e x v i r u s t y p e 1 ( r e f . 102); herpes s a i m i r i v i r u s ( r e f . 99); and SV40 ( r e f . 101) do n o t c o n t a i n d e t e c t a b l e m o d i f i e d bases d e s p i t e m5C b e i n g p r e s e n t i n t h e i r h o s t DNAs. However, i n 10.4
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tegration of a number of the above DNAs as well as of retroviral provi ral DNA into host genomes in a transcriptional ly repressed form (but not in the transcriptionally active form) is associated with extensive DNA methylation to give m5CpG sites (refs. 104, 105, 106, 107, 108, 109). Although i n f e c t i o u s viral eukaryotic DNAs seem to be generally unmethylated, several exceptions have been reported. DNA from virions of frog virus 3 contains considerable amounts of m5C (ref. 110). Also, viral extrachromosomal DNA from another member of the iridovirus virus family, fish lymphocytosis disease virus, is methylated at 75% of its CpG dinucleotides when isolated from infected tissues (ref. 111). Evidence has been presented for a very slight degree of CpG methyl ation of extrachromosomal human papi 1 loma viral DNA isolated from warts (ref. 112). The extrachromosomal viral DNA in Shope papi 1 loma vi rus-i nfected neoplasms is more highly, but variably, methylated at CpG sites (ref. 113). The only report of a eukaryotic virus associated with a DNA modification other than m5C is a virus called NC-lA, which infects a chlorella-like green alga (ref. 114). This viral DNA contains mA in 5'-GANTC-3' sequences assoc ated with a restriction-modification type system. Its DNA has a high level of m5C (7 mol%) as well as of m6A (7 mol%). Analyses of organelle DNA indicate little or no methylation Chloroplasts from tobacco leaves were reported to contain no detectable m5C in their DNA although the detection limits in this analysis were not indicated (ref. 115). Mitochondria1 DNA from Paramecium a u r e l i a contains (0.1 mol% m5C or m6A; the nuclear DNA also has no detectable m5C but has 2.5 mol% m6A (ref. 79). Less than 0.1 and 0.05 mol% m5C was detectable in the mitochondrial DNA of frog and cultured human (HeLa) cells, respectively (ref. 116). Examination of mitochondria1 DNA from various cultured mammalian cell lines indicated that it contains one-fourth to onefourteenth the m5C content of nuclear DNA (ref. 117). Similarly, we found a low level of m5C (0.14 mol%) in DNA from a mitochondrial fraction of human placenta (ref. 118). Restriction analysis of this DNA indicated (5% contamination with bulk nuclear DNA. However, in these studies of organelle DNA, covalently closed circular DNA is isolated from partially purified subcellular fractions which might be contaminated with small circular DNAs.
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These may c o n t a i n c e r t a i n n u c l e a r DNA sequences t h e r e b y i n t r o ducing contaminating m5C r e s i d u e s . In t h i s regard r e s t r i c t i o n a n a l y s i s of t h e s e o r g a n e l l e DNAs i s l e s s l i a b l e t o f a l s e p o s i t i v e r e s u l t s . We found no d e t e c t a b l e methylation a t 5'-CCGG-3' s i t e s i n human p l a c e n t a l DNA by comparing HpaII and MspI d i g e s t s ( r e f . 119). S i m i l a r l y , no evidence f o r CpG methylation of wheat, y e a s t , Neurospora, r a t , o r c a l f mitochondria1 DNA was found by r e s t r i c t i o n a n a l y s e s ( r e f s . 120, 121). In c o n t r a s t , the u n i c e l l u l a r green a l g a Chlamydomonas methylates almost h a l f o f t h e C r e s i d u e s i n i t s c h l o r o p l a s t DNA i n t h e m a t e r n a l l y derived b u t n o t the p a t e r n a l l y derived c h l o r o p l a s t DNA i n zygotes ( r e f s . 122, 123, 1 2 4 ) . The b i o l o g i c a l impact of t h i s methylation i s u n c l e a r . D I S T R I B U T I O N OF m5C I N THE NUCLEAR DNA OF HIGHER PLANTS AND VERTEBRATES All studied higher p l a n t s c o n t a i n m5C a s a minor b u t cons i d e r a b l e c o n s t i t u e n t of t h e i r n u c l e a r DNA ( 2.3-7.1 mol%) just a s a l l v e r t e b r a t e genomes appear t o have m5C ( 0.7-2.8 mol%) a s t h e i r only minor base ( r e f s . 1, 1 2 5 ) . P l a n t DNAs can have up t o 33% of 10.5
t h e i r C r e s i d u e s methylated (tobacco: r e f . 125) and even higher percentages of the C r e s i d u e s i n c e r t a i n s a t e l l i t e DNAs (melon: r e f . 126; b l u e b e l l : r e f . 127). The d i s t r i b u t i o n o f m5C r e s i d u e s i s sequence-specific ( r e f . 128) with methylation o c c u r r i n g predominately a t CpG o r CpNpG (N i s any base) sequences ( r e f s . 129, 127, 130, 131). Although p l a n t DNA methylation has been s t u d i e d r e l a t i v e l y l i t t l e , there i s a t l e a s t one example of t i s s u e - s p e c i f i c d i f f e r e n c e s i n p l a n t DNA methylation ( r e f . 1 3 2 ) . This observed t i s s u e - s p e c i f i c change i n DNA methylation o f t h e z e i n gene i n d i c a t e s t h a t gene demethylation might, i n some c a s e s , p o s i t i v e l y c o n t r o l gene expression ( r e f . 132). A s t u d y of i n vivo methylation of the transforming plasmid fragment (T-DNA) from Agrobacterium tumefaciens, a b a c t e r i um oncogeni c f o r d i c o t y l donous p l a n t s , s u g g e s t s t h a t methylation of T-DNA i n s i d e p l a n t c e l l s might r e g u l a t e i t s t r a n s c r i p t i o n ( r e f . 133). Another proposed r o l e f o r p l a n t DNA methylation which has experimental s u p p o r t i s t h a t i t might d e c r e a s e t r a n s p o s i t i o n by t r a n s p o s a b l e elements r e s i d e n t i n p l a n t nuclei ( r e f . 134). In c o n s i d e r i n g t h e r o l e s of DNA m e t h y l a t i o n , the mode of f o r -
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mation of m5C residues should be evaluated. Methylation is catalyzed by a cytosine DNA methyl transferase with S-adenosylmethionine and DNA as the substrates after DNA replication (refs. 135-137). The methyl atable ol igonucleotide sequences (CpG and CpNpG) have dyad symmetry, that is, in the 5' ---> 3' direction the same sequence is present on both strands. This facilitates the recognition of plant DNA methylated in only one strand so that such hemimethylated DNA serves as the best DNA substrate for the plant's DNA methyl transferase(s) enzyme(s) (ref. 137). Methylation of hemimethylated DNA is referred to as maintenance methylation because it serves to conserve the pattern of DNA methylation that existed before DNA replication converted a bifilarly (symmetrically) methylated to a hemimethylated site (ref. 1, 127). I n contrast, increases in net DNA methylation that can help establish tissue-specific patterns of DNA methylation or genetic polymorphisms in the population are catalyzed in a de n o w methylation reaction, that is, i n a reaction utilizing a bifilarly unmethylated DNA sequence as the substrate. Similar considerations pertain to vertebrate DNA methylation and vertebrate DNA methyltransferases except that i n this case the site of methylation is predominantly or only CpG (ref. 1). Vertebrate DNAs contain only approximately one-fourth the expected frequency of CpG dinucleotide (ref. 138). Because the majority of CpG sites are methylated and CpG in the predominant site of vertebrate DNA methylation (refs. 2, 104, 139), the limitation on the frequency of mCpG sites is postulated to be due to the accumulation of m5C ---> T transition mutations (refs. 140-144). Both m5C and C residues can deaminate in a heat-induced reaction which should also occur, although to a lower extent, at physiological temperature (refs. 32, 33). Deamination o f an m5C residue yields a T residue which is a normal constituent of DNA unlike the C deamination product, U, which should be efficiently excised from DNA (refs. 32, 145). Furthermore, heat-induced deamination of m5C residues in single-stranded DNA in a physiological buffer occurs at a faster rate than for the analogous C residues (refs. 146, 33). For vertebrates, the extent and pattern of DNA methylation has been shown in many studies to be tissue-specific although, i n
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all cases, CpG dinucleotides appear to be the predominant site of methylation (refs. 1, 147-150, 1 5 2 ) . Generally, the majority of CpG dinucleotides in vertebrate DNA are methylated a1 though the percentage o f unmethylated CpG sites is considerable and varies from tissue to tissue (refs. 1, 150). Not only can the level of DNA methylation differ as much as 30% from one tissue to another and the patterns of DNA methylation vary much within normal cells of an organism, but also, as described below, the amount and distribution of genomic m5C can be highly altered upon tumorigenesis or oncogenic transformation (refs. 153-155). A1 though m5C is generally enriched in heterochromatin-associated DNA in vertebrates, much of it is also in single copy, moderately repeated, and interspersed repeated DNA fractions (refs. 156, 1 5 0 ) . The distribution of unmethylated CpG dinucleotides also shows a tendency toward clustering (refs. 157-159). Some DNA subfractions, like the DNA sequences encoding ribosomal RNA (rDNA) are either unusually m5C-rich (rDNA in fish and amphibi a) or m5C-poor (rDNA i n mammal s , repti 1 es, and bi rds) depending on the type of organism (ref. 141). The methylation status of a DNA sequence can change greatly depending upon whether it is chromosomal or extrachromosomal (refs. 150, 161) or upon its chromosomal location (ref. 139). THE FUNCTIONAL SIGNIFICANCE OF VERTEBRATE DNA METHYLATION: TRANSCRIPTION, CHROMATIN STRUCTURE, DNA REPLICATION AND REPAIR, CANCER AND EMBRYOGENESIS Vertebrate DNA methylation appears to be involved in controlling expression of certain genes. There are many examples of correlations of naturally occurring DNA methyl at ion and the inhibition of gene expression in mammalian and avian genomes (refs. 162-170; and reviewed in ref. 1; 172, 173). In a number of cases it is clear that genetically programmed, decreases in methylation (demethylation) of DNA sequences cannot be sufficient to activate gene expression although they might be necessary for turning on transcription (ref. 174-177). A1 though the mechanisms for methylation-directed transcription control remain to be established, two types of studies have provided insight into this phenomenon. In the first of these, evidence has been presented 10.6
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that a high density of m5C residues affects how DNA is packaged in nucl eosomes and supranucleosome particles (ref. 178, 179). In the second line of experimentation, a DNA-binding protein has been i sol ated from human pl acental nuclei , whi ch speci fi cal ly binds to certain 20-base-pai r DNA sequences only when they are methyl ated at their 2 or 3 CpG sites (refs. 180-183). The high degree of sequence-speci fi ci ty and methyl ati on-dependence of this bi ndi ng reaction suggests that this protein, methylated DNA-binding protein (MDBP), might be a novel transcription regulatory protein specifically responsive to local DNA methylation. Some of the most striking correlations between DNA methylation and silencing of gene expression have been made for mammalian and avian viral genomes or for proviral DNA integrated into mammal i an genomes where they can become methyl ated (refs. 105, 184-192). In some cases, the lack of DNA methylation was shown not to be sufficient for transcriptional activity although evidence suggests that de novo methylation is part of the mechanism for turning off viral genes in the host genome or for keeping them inactive (refs. 193-195). I n v i t r o DNA methylation of a viral promoter (refs. 196, 197) or of the whole proviral DNA (ref. 198) eliminated most or all o f its activity. In one case, methylation caused a shutdown in expression only after a prolonged delay (ref. 199). Demethylation of viral DNAs by 5-azacytidine, which is a pleiotropic antimetabolite that is the strongest known inhibitor of i n v i v o DNA methylation (refs. 200, 201), activates the expression of many viral DNA genomes that have become methylated upon integration into the host genome (refs. 106, 202-205). Similarly, 5-azacytidine or 5-aza-Zt-deoxycytidine treatment turned on expression of many vertebrate genes and induced differentiation in cultured cells (refs. 206-218). Furthermore, d e n o v o methylation of the human 7-globin promoter region prevented its transcription upon introduction into mouse L eel Is, whereas methylation of most of its protein-coding DNA sequence did not (ref. 219). This suggests that methylation at many sites in genes has no effect upon gene expression but at other sites does. This is consistent with our finding that methylation changes during the course of human development at >10 x 106 sites per haploid genome (ref. 150), an extent of change far too great for much of it to be
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invo ved directly in controlling gene expression. Similar results have been found for other mammals (ref. 220). As these results pred ct, a number of tissue-specific differences i n DNA methylation patterns have been found which cannot be correlated with transcriptional activity (refs. 174, 221-223) or which may be consequences rather than causes of transcriptional activation (refs. 224, 225). There are several lines of evidence suggesting that vertebrate DNA methylation plays an important role in determining the structure of chromatin. Satellite DNAs from mammalian tissues, which are found in highly condensed chromatin (heterochromatin), often have a higher m5C content than the low-copy fraction of DNA (refs. 150, 151, 226-230). Heterochromatin is often associated with centromeres. By analysis for immunoreactive sites on metaphase chromosomes from various mammals, m5C residues have been found to be highly enriched in centromeric regions (refs. 231, 232). The distribution of such immunoreactive m5C sites changes during meiosis (ref. 233). Treatment of cell s with 5-azacytidine induces decondensation of heterochromatin (refs. 234-236) as well as the formation of fragile sites and sister-chromatid exchanges (refs. 235-238). In the mealybug, a high mC content in male DNA was correlated with the presence of a paternally derived, genetically inactive facultative heterochromatin (ref. 96). That the methylation status of satellite DNA is physiologically important is suggested by the much higher level of methylation o f several mammalian satellite DNAs in embryoblast (primitive ectoderm-derived) and adult somatic ti ssues than in the extraembryonic trophobl ast and primitive endoderm derivatives or in gametes (refs. 151, 239-241). Not only has DNA methylation been implicated in higher order chromatin structure, but also, m5C residues have been shown to be preferentially associated with nucleosome core, histone H1-associated chromatin (refs. 178, 242). In addition, DNA cytosine methylation can have a large influence on the conformation of the DNA helix, such as, favoring the Z-DNA conformation (ref. 243). These findings suggest that DNA methylation critically influences the condensation of DNA i n chromatin and of certain satellite DNAs in heterochromatin and also may
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subtly affect local chromatin structure. As mentioned above, this, in turn, could be related to the transcriptional activity of DNA and, also, it could affect the ability of chromatin to undergo various types o f recombi nation. DNA methylation has been linked to another form of chromatin condensation namely, the heterochromatinization and transcriptional inactivation of one of the two X chromosome in female mammals. Much evidence suggests that DNA methylation is intimately involved i n this phenomenon (refs. 244-248, 169). Similarly, DNA methylation has been implicated in the late timing of replication of the inactive X chromosome during S phase (refs. 249, 250, 251). It is also possible that DNA methylation plays a role in controlling the initiation of DNA replication as it appears to in Escherichia c o l i (ref. 72). A n additional analogy to bacterial DNA is suggested by evidence that hemimethylation (at C residues) of CpG sites directs mismatch repair in mammalian cells (ref. 252) as does hemimethylation (at A residues of 5'-GATC-3' sites) i n E . c o l i (ref. 75). As described above, there are extensive tissue-specific differences in the amount and distribution of m5C in vertebrate DNA and some of these, probably only a small percentage, are involved i n controlling gene expression. This indicates that DNA methylation probably plays a major role i n guiding differentiation. It is informative, therefore, to compare the methylation status of specific DNA sequences in gametes and somatic cells. A number of vertebrate genes have been shown to be much more methylated at tested CpG sites in sperm DNA than in one or more adult somatic tissue DNAs (refs. 168, 173, 221, 253-255). On the other hand, human sperm DNA has a rather low overall m5C content compared to somatic tissues, which seems to be due to hypomethylation of satellite DNA sequences, as mentioned above, (ref. 220) as well as to hypomethylation of non-satellite (including low-copy-number) sequences which are speci fical ly undermethyl ated in sperm (refs. 158, 159) and probably also i n oocytes (ref. 240). The latter DNA sequences show similar hypermethylation at both 5'-CCGG-3' and 5'-GCGC-3' sites over a long region in all human adult somatic tissues i n contrast to their hypomethyl ation in sperm and intermediate level of methylation in placenta (refs.
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158, 159). The unusual m e t h y l a t i o n p a t t e r n i n t h e s e sequences i n p l a c e n t a i s c o n s i s t e n t w i t h i t s v e r y d i s t i n c t c e l l l i n e a g e and i t s DNA's r a t h e r low o v e r a l l m5C c o n t e n t ( r e f s . 150, 256). The r e s u l t s w i t h sperm DNA suggest t h a t hypomethylation o f many d i s p a r a t e DNA sequences may be necessary f o r gametogenesis o r e a r l y embryogenesis. T h i s may be f o l l o w e d by an i n c r e a s e i n m e t h y l a t i o n o f these sequences e a r l y i n development y i e l d i n g s i m i l a r hypermethylation i n a l l tissues destined t o g i v e r i s e t o the adult. I n a d d i t i o n t o these DNA sequences hypomethylated s p e c i f i c a l l y i n gametes, t h e r e a r e o t h e r a t y p i c a l l y undermethylated DNA sequences which appear t o be undermethylated i n a77 c e l l populat i o n s o f an organism. They a r e sometimes found i n 5 ' gene r e g i o n s n e x t t o a h i g h l y m e t h y l a t e d r e g i o n w i t h i n t h e gene ( r e f . 157). T h i s asymmetri c a l p l acement o f m5CpG s i t e s a1 ong t h e chromosome m i g h t be one s i g n a l f o r i d e n t i f i c a t i o n o f c e r t a i n genes as t r a n scriptional units. A s d e s c r i b e d above, DNA m e t h y l a t i o n i n v e r t e b r a t e s may h e l p i n d i v e r s e ways t o r e g u l a t e macromolecular s y n t h e s i s and d i f f e r e n t i a t i o n . Therefore, derangements i n normal DNA m e t h y l a t i o n c o u l d have p l e i o t r o p i c consequences t o c e l l u l a r p h y s i o l ogy. Indeed, t h e r e i s evidence suggesting t h a t a b n o r m a l i t i e s i n DNA m e t h y l a t i o n a r e i n v o l v e d i n carcinogenesis. Some o f t h i s evidence comes from a n a l y s i s o f d e m e t h y l a t i o n caused b y c a r c i n o g e n i c agents ( r e f s . 165, 205, 257-264). Furthermore, many o f t h e v i r a l DNAs and p r o v i r u s e s whose expression can be c o n t r o l l e d by DNA m e t h y l a t i o n a r e oncogenic v i r u s e s ( r e f s . 106, 107, 185, 187, 196-198, 202, 205). A l s o , s t u d i e s o f human tumors, w i t h o u t t h e p o s s i b l y comp l i c a t i n g intermediacy o f c e l l c u l t u r e , i n d i c a t e a r e l a t i o n s h i p between a1 t e r a t i o n s i n DNA m e t h y l a t i o n and c a r c i n o g e n e s i s . Examination of 23 human c o l o n neoplasms and normal a d j a c e n t i n t e s t i n a l mucosa by Feinberg, V o g e l s t e i n and coworkers ( r e f s . 154, 265, 266) r e v e a l e d t h a t some d i v e r s e genes have a h i g h probabi 1 it y o f becomi ng hypomethyl a t e d d u r i n g f o r m a t i o n o f c o l o n tumors. T h i s was seen i n genes l i k e t h a t o f 7-globin, whose expression ought t o be i r r e l e v a n t t o t h e tumor as w e l l as i n an oncogene, whose m e t h y l a t i o n m i g h t o r m i g h t n o t be o f p h y s i o l o g i c a l s i g n i f i c a n c e i n t h i s tumorigenesis ( r e f s . 154, 266). The hypo-
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methylation of these genes showed much heterogeneity i n its pattern but clearly was not random. In a collaborative study involving C. Gehrke, K. Kuo, A . Feinberg, and one of us (M. Ehrl ich), we have recently demonstrated that the demethylation n these colon tumors, which are known to arise from the essential Y homogeneous cell population of intestinal mucosa, is part of a genome-wide demethylation (ref. 155). Simi 1 arly, chemical ly induced hepatocarcinomas in rats had a lower m5C content than did normal rat liver (ref. 267). Furthe more, despite a wide range of genomic m5C contents, (0.35-1.03% mol% m5C), a statistically significant difference was found in the distribution of DNA methylation levels in various human malignancies, especially in metastases, as compared to that in benign neoplasms or normal human tissues (ref. 153). The percentages of DNA samples with <0.80 mol% m5C or >0.84 mol% m5C were strikingly different for malignancies and for normal tissues or benign tumors (ref. 153). These differences suggest that either hypomethylation often accompanied tumorigenesis or that many of these malignancies were derived from atypical, minor populations of cells with relatively low m5C contents. In the study o f colon tumors mentioned above, we can conclude that decreases in DNA methylation occurred because the tumors are known to arise from the predominant cell type of intestinal mucosa and methylation of tumor DNA was compared to that of the adjacent normal mucosa (ref. 155). The m5C content of neoplastic samples should reflect the percentage of neoplastic cells in the sample, the type of cells which gave rise to the neoplasm, and any changes in DNA methylation which occur during the early stages of oncogenic transformation or during tumor progression. We have proposed that tumor progression with its attendant continually generated cell ul ar diversity is often accompanied by extensive replacement of m5C residues in DNA with cytosine residues (ref, 153). Studies of cultured murine cell s and transplantable tumors support this hypothesis (refs. 260, 261, 268). Hypomethylation of DNA during tumor progression could provide epigenetic changes of the type associated with normal differentiation. It could help establish or mai ntai n transcriptional activity or 1 ead to cancer-re1ated chromosomal rearrangements, gene amplification, and a1 terations in
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chromosome conformation. The resulting changes in expression or replication of the genome might be an important component in the diversification of tumor cells, which allows them to successfully exploit thei r hosts. 10.7 SUMMARY
Most DNAs contain minor amounts of 5-methylcytosine (rn5C), N6-methyladeni ne (m6A), or N4-methyl cytosine (in"). Any one or more of these can be found in most bacterial DNAs. Some lower eukaryotes have m5C, m6A or both as a minor base in their genomes. In the DNA of all studied vertebrates and higher plants, m5C, and only m5C, is the naturally occurring modified base. The levels and patterns of m5C in vertebrate genomes are ti ssue-speci fi c. Some, but clearly not most, vertebrate DNA methylation has been linked to the regulation of gene transcription. As for m6A in the Escherichia c o l i genome, m5C in the DNA of eukaryotes may function in controlling DNA repair, replication, and rearrangements as well as in control of transcription. However, although bacterial DNA methylation is often involved in r e s t r i c t i o n - m o d i f i c a t i o n systems, there is no evidence for this function in the case of vertebrate DNA methylation. Vertebrate DNA methylation may, however, be an important determinant of chromatin structure. In addition to methylated bases present as minor DNA components, other modified bases are major components of certain bacteriophage genomes. These have one of their bases, usually a pyrimidine, largely or completely replaced by the modified derivative. The derivatives include gl ucosyl ated 5-hydroxy-methylcytosine, uracil, 5-hydroxymethyluracil, 5-(4-aminobutylaminomethy1)uraci 1 , and gl ucosyl ated and gl ucuroni c aci d-l-phosphorylated 5-(4',5'-dihydroxypenty1)uracil. These major modifications of phage genomes appear to help, sometimes in subtle ways, the virus to distinguish its genome from that of the host in its exploitation of the host cell and to evade host DNA restriction systems . 10.8
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