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Single-strand interactions of mouse satellite DNA
BIOCHIMICA ET BIOPHYSICA ACTA
297
BBA 97136
S I N G L E - S T R A N D I N T E R A C T I O N S OF MOUSE S A T E L L I T E DNA D A V I D M. K I ; R N I T , C A R L L. S C H I L D I ( R A U T AND J O S E P H J. ~.rAIO
Division o[ Biologica/ .q'ciences (Cell Biology), .4lbert Einstein Cv/lege o/ 3Iedicinc, t3ronx, N.3". Lo46~ (U.S..-I.) (Received A u g u s t 24th, 1971)
SUMMARY
Interactions of the complementary strands of mouse satellite DNA were investigated in the presence of polyribonucleotides, at high ionic strengths, and at different molar ratios of the two strands. Polyribonucleotide binding demonstrated the presence of T-rich and (A,C)-rich or C-rich sequences on the heavy strand. Ribopolymers bound to one strand of mouse satellite DNA were displaced by addition of the complementary DNA strand. However, the formation of mouse satellite DNA • DNA hybrids is essentially irreversible. High ionic strengths and formaldehyde were used to investigate the two steps, nucleation and rewinding, which comprise the reassociation of mouse satellite DNA. Compared with procaryotic DNA's, the complementary strands of mouse satellite DNA nucleate rapidly. The rewinding of nucleated complementary strands of mouse satellite DNA is inhibited at high ionic strengths and low temperatures. The effects of rapid nucleation of mismatched strands on the reassociation of animal satellite DNA's are discussed.
INTRODUCTION
Unlike most naturally-occurring DNA's, the buoyant density and melting temperature of mouse satellite DNA do not correspond to the values predicted from its base composition 1-3. In addition, the separated strands reassociate rapidly 4 to form a mismatched hybrid 1,2. These properties are due to the nucleotide organization of mouse satellite DNA, which apparently contains repeats of (and variations on) a homopolymer-like sequence, TTTTTC ~. This report describes the interactions of the complementary strands of mouse satellite DNA in buffers of various ionic strengths in the presence of polyribonucleotides, and at different ratios of the two strands. The results demonstrate a correlation between the nucleotide organization of mouse satellite DNA and its reassociation properties.
METHODS AND MATERIALS
Nuclear DNA from a mouse neuroblastoma tissue-culture line with a molecular weight of approx, lO 7 was obtained by a modification of MARMUR'S3'6 method. Mouse satellite DNA was isolated by preparative density-gradient centrifugation in Biochim. Biophys. ~4cta, 259 (I972) 297-312
1). M. KURNIT t'[ al.
298
A g ÷ Cs2SO 4 (ref. 2} d i a l y z e d a g a i n s t 2 M NaC1 in T r i s - E D T A b u f f e r (IO m M Tris HC1 plus I m M E D T A (pH 8.4) ), a n d t h e n d i a l y z e d a g a i n s t T r i s - E D T A buffer. T h e c o m p l e n l e n t a r y h e a v y (H) a n d ligllt (L) s t r a n d s of m o u s e s a t e l l i t e D N A were sep a r a t e d b y c e n t r i f u g a t i o n in p r e p a r a t i v e a l k a l i n e Cs('l (pH 12.5) density" g r a d i e n t s 7. T h e s e p a r a t e d s t r a n d s w e r e t h e n d i a l y z e d a g a i n s t T r i s - E D T A buffer. TAI3LE 1 BUOYANT
DENSITIES
OF ANIMAL
SATELLITE
[)NA's
Buoyant density and base composition data for the duplex satellite DNA's and their complementary strands were obtained from the sources listed under Re/erence. Buoyant densities of the duplex molecules in CsC1 were calculated from their base compositions according to the equation: f, ~- o.o9S ((; t C)+ ~.659 {~) where p is the DNA buoyant density in g/cm a and (G ! C) is the average mole fraction of Rimnine and cytosine ~2. ]3uoyant densities of the single-stranded molecules in CsC1 at pH 8. 4 were calculated from their base compositions according to the equation: p - - 1.62(~69A-}-1.758o4G~ ~.768o9 C ~ 1.7424IT {2) where p is the DNA buoyant density in g/cm a and A, G, C and T are the average mole fractious of each of the four bases 9. The density of E. coli native DNA was taken as 1.71o g/cm 3 in all tmoyant density determinations.
Satellite 1)XA
Re/erence
Mouse satellite
2,3,7; Table I1 this paper
Duplex L strand H strand Guinea pig satellite I Duplex I. strand H strand Human satellite [ Duplex 1. strand H strand
Observed buoya~z! density (g/cm 3)
Buoyant densitiy (g/cma)/rom Eq~2. r (double stranded DN.4)
1.689 i .699 i .728
1.695
1.7o 5 1.7o6 .754
i .608
i .686 1.694 1.712
~.685
Buoyant dealsity (g/cma)#om Eqn. 2 (singh,stranded DNA )
[ .608 t.727
53 i. 709 t.724
23, 12 ---
i .696 1.712
A n a l y t i c a l d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n in a S p i n c o M o d e l E u l t r a c e n t r i f u g e w a s as d e s c r i b e d p r e v i o u s l y a. I n n e u t r a l CsC1, D N A f r o m t h e Bacillus subtilis b a c t e r i o p h a g e 6C (density, 1.742 g / c i n a) a n d p o l y E d ( A - T ) -d(A-T)I~ (density, 1.679 g / c m a) w e r e u s e d as s t a n d a r d s for d e n s i t y d e t e r m i n a t i o n . T h e s e v a l u e s c o r r e s p o n d to a d e n s i t y of 1.71o g / e m 3 for Escherichia coli D N A . I n a l k a l i n e CsCl (pH I2.5), t h e h e a v y s t r a n d of t h e B. subtilis b a c t e r i o p h a g e 6C D N A was t a k e n to be 1.8Ol g/cin "~, c o r r e s p o n d i n g to a v a l u e of 1.772 g / c m a for E. coli D N A c e n t r i f u g e d u n d e r similar c o n d i t i o n s . F o r e e n t r i f u g a t i o n in t h e p r e s e n c e of f o r m a l d e h y d e , t h e D N A was d i a l y z e d a g a i n s t b o r a t e b u f f e r (5 inM N a 2 B 4 0 7 p l u s 5 m M Na2SO 4 (pH 8.8)). T h e a n a l y t i c a l u l t r a c e n t r i f u g a t i o n was in CsC1 d i s s o l v e d in b o r a t e buffer. T h e f o r m a l d e h y d e was d i l u t e d f r o m a 4 ° °~b s t o c k s o l u t i o n of f o r m a l i n (Merck).
Biochim. Biophys. Acta, 259 (i972) 297-312
MOUSE SATELLITE DNA
299
Polyribonucleotide binding The preparation and calibration of the stock solutions of polyribonucleotides were described in an earlier paper s. Polyribonucleotides were added to DNA in T r i s - E D T A buffer to the desired ratio of polyribonucleotide phosphorus to DNA phosphorus in a total volume of IiO/~1. Unless otherwise stated, this ratio was I : I or 2 : 1 . Initially, experiments were performed by heating tile native or singlestranded DNA samples in T r i s - E D T A buffer at 9 °o for 5 rain in the presence of polyribonucleotides. It was later found that heating at 9 °o was not necessary for binding to the single strands. The same results were obtained when tile polyribonucleotides were added directly to the DNA in Tris EDTA buffer or in CsC1 at 25 °.
RESULTS AND INTERPRETATION
Properties o/the isolated single strands The densities in CsC1 (pH 8.4) of the complementary strands of mouse satellite DNA were 1.728 g/cm a and 1.699 g/em a, in agreement with previously published values (ref. 2 and Table II). The bands formed in CsC1 were sharp and symmetrical, with no shoulders. The buoyant densities of the single strands coincided with the densities calculated from the base composition of each strand by means of an empirical equation which relates the base composition and buoyant densities of singlestranded DNA's (ref. 9 and Table I). Interestingly, this equation applies to all of tile single strands of the satellite DNA's listed in Table I with the exception of the H strand of guinea pig satellite I DNA. TABLE BUOYANT
II DENSITIES
OF MOUSE SATELLITE
DUPLEX
AND SINGLE-STRANDED
1)*~N~A
Expt. No.
Sample
Duplex
H strand
L strand
I 2
D u p l e x i n CsC1 ( p H 8.4) D u p l e x in T r i s E D T A b u f f e r , 5 / * g / m l , h e a t e d a t 9 °o f o r 5 rain and fast cooled D u p l e x in CsC1 ( p H 12.5) S e p a r a t e d s t r a n d i n CsCl ( p H 8.4) S e p a r a t e d s t r a n d in T r i s E D T A b u f f e r , 5 ~¢g/ml, h e a t e d at 9 °° for 5 min S e p a r a t e d s t r a n d in o. 3 M N a C I , 5 t l g / m l , heated at 60 ° for i h
1.689
--
--
1.71o --
-1.767
-1.742
---
1.728
1.699
--
1.728
1.699
--
1.729
1.699
3 4 5
6
As measured by hydroxyapatite chromatography and absorbance, a significant amount of intrastrand folding occurs when either H or L strands are self-annealed 1°. However, changes in intrastrand folding introduced by denaturing or annealing single strands before density gradient centrifugation did not affect the final buoyant densities (Table II). The high ionic strength of the 7.9 molal CsC1 solution enhances the stability of hydrogen bonds and increases intrastrand folding n. It is probable Biochim. Biophys. Acta, 2 5 9 ( 1 9 7 2 ) 2 9 7 - 3 1 2
300
D.M. KURNIT e[ al.
t h a t the high degree of i n t r a s t r a n d folding of s i n g l e - s t r a n d e d D N A ' s in c o n c e n t r a t e d CsC1 solutions m a s k s pre-existing differences in i n t r a s t r a n d h y d r o g e n bonding. F r a c t i o n s from the light a n d h e a v y sides of each single s t r a n d isolated in the p r e p a r a t i v e alkaline CsC1 d e n s i t y g r a d i e n t were rerun in a n a l y t i c a l alkaline CsC1 d e n s i t y gradients. H s t r a n d fractions b a n d e d from L768 to L765 g/cma; L s t r a n d fractions r a n g e d from 1.743 to 1.741 g/cm :3. The reassociation characteristics of these fractions confirmed t h a t c o n t a n f i n a t i n g m a i n b a n d D N A was not present. Thus, a l t h o u g h mouse satellite D N A is more homogeneous t h a n m a i n b a n d D N A t2, d e n s i t y h e t e r o g e n e i t y was detectable. H e t e r o g e n e i t y in the basic nucleotide sequence of mouse satellite D N A has been o b s e r v e d p r e v i o u s l y 5. l~olvribonucleotide binding H e a t - d e n a t u r e d m a i n b a n d D N A b o u n d p o l y ( U ) , poly(G), a n d p o l y ( U , G), a n d showed the b u o y a n t d e n s i t y increase which is c h a r a c t e r i s t i c of a n i m a l D N A ' s ~3 (Table In). The single b r o a d p e a k o b t a i n e d was due p r e s m n a b l y to d e n s i t y heterog e n e i t y a n d to b i n d i n g of a p p r o x i m a t e l y equal a m o u n t s of p o l y r i b o n u c l e o t i d e s b y b o t h s t r a n d s of t h e D N A molecules. W h e n d u p l e x satellite D N A was r e a c t e d w i t h t h e p o l y r i b o n u c l e o t i d e s u n d e r similar conditions, n m c h less b i n d i n g was d e t e c t e d . However, the D N A b a n d e d at a b o u t the d e n a t u r e d density. In contrast, the s e p a r a t e d
TAI~LE II[ POLYRIBONIJCL],;OT1DFBINDINGTO rqOUSl"I)~N~.\ Sample
Polyribom~cleolide (s) added
Huoyant dcnsilv (g/cm 3)
Mouse main band, 25' MZouse nlain band, 9 0 5 lnill
]N*Olle
1.7oo
.'NTOile Poly(IT) Poly(G) Poly(l',G)
~.715 r.729 1.726 1.756
None
1.689
~-N'OIIe Poly(U) Poly(G) Poly(IT,G}
1.71o 1.7I 4 1.71o 1.715
~Olle Poly(U) Poly(G) Poly(U,G)
.699 1.788 1.698 1.7o8
None Poly(U) Poly(G) Poly(U,Z~) Poly(t',G) ] Poly(G)
1.728 1.727 1.742 1.779 1.773
NIouse satellite, duplex, 2 5 5'[ouse satellite, duplex, 9o', 5 rain
Mouse satellite, separated L strand
Mouse satellite, separated H strand
Biochim. Iliophys..-tcta, 259 (i972 ) 297-312
Huoyanl density increment over denatured de~lsity (mg/cm~)
o 14
ii 4 r
89 o 9
o
14 5I 45
MOUSE SATELLITE
DNA
3Ol
strands of mouse satellite DNA bound polyribonucleotides in a highly specific and asymmetric pattern. Poly(U) bound only to the L strand, and resulted in a buoyant density increment of 89 mg/cm 3 (Table I I I ) . This was the largest density increment we observed with this technique in any animal or microbial DNA. The asymmetric pattern was further demonstrated by the selective binding of poly(U,G) and poly(G) to the H strand. The binding sites for poly(U, G) and poly(G) apparently coincide, since the addition of both ribopolymers did not have an additive effect on the buoyant density increment of the H strand. Complexes between the separated DNA strands and polyribonucleotides formed readily at 25 ° in 7-9 molal CsC1. Poly(A) and poly (A,C) did not bind, perhaps because of specific conformational effects. We conclude that mouse satellite DNA H strand contains extensive regions of T-rich sequences as evidenced by the large amount of poly(U) binding by the L strand. This is consistent with pyrimidine tract data which suggest that TTTTTC is included in the basic sequence s. The presence of poly(U,G) and poly(G) binding sites on the H strand suggests that there is another repeated sequence, or that (A,C)-rich (or C-rich) clusters fill out the remainder of the basic sequence which is postulated to be 8-13 nucleotides long 5. Since (A,C)-rich sequences are disrupted by diphenylamine-formic acid hydrolysis 14, this sequence would not be detected by pyrimidine tract analysis. Main band DNA contains ribopolymer binding sites as does satellite DNA (Table I I I ) ; it is possible that these sequences account for the limited binding of satellite DNA to main band DNA 1°. Asymmetric polyribonucleotide binding has been observed in bacterial and bacteriophage DNA's 1~,15, and in a rapidly reassociating fraction of African green monkey DNA s. In bacteriophages, the presence of pyrimidine-rich sequences on one strand has been associated with preferential transcription of that strand 15. By this criterion, only the H strand of mouse satellite DNA would be transcribed. However it is not clear whether mouse satellite DNA is transcribed in vivo 1°'16. The e//ects o/ ionic strength on reassociation Renaturation of nucleic acids appears to be a two-step process 17,18. The first step, nucleation, is a bimolecular reaction involving the collision and complementary pairing of a limited number of base pairs. The smallest number of such base pairs forming a duplex region stable enough to permit further helical growth is termed the stable minimum length 19. The second-order rate constant of the nucleation reaction depends directly upon the number of complementary nucleotides on the interacting strands long enough to function as a stable minimum length. Since the stable minim u m length need be only about 12-3o nucleotides long 2°-22, nucleation is a measure of the homology of short nucleotide sequences on the interacting molecules. The second step, rewinding, is a unimolecular reaction in which helical growth follows successful nucleation. This reaction also varies with the base sequence homology of the interacting DNA's, but in a more complex way than the nucleation reaction. When mouse satellite DNA H and L strands were mixed and centrifuged in an analytical CsC1 density gradient (7.9 molal CsC1, 20 h, 25°), one band at a density of 1.709 g/cm 3 was observed (Table IV). The density of this band was 0.02 g/cm 3 heavier than the native density of 1.689 g/cm ~. This density increase is characteristic of denatured DNA. The presence of only one band at the denatured DNA density indicated that nucleation had occurred without significant rewinding. Under these Biochim. Biophys..4eta, 259 (1972) 297-312
302 TABLE
1). M. K U R N I T e~ ( d . IV
FORMALDEHYDE
Expt. No.
REACTION
~,VITtt S E P A R A T E D
STRANDS OF MOUSE SA.TELLITE ])N ,\
1 nc~tbation conditions
Number o/ I)N.4 bands observed
1~uoya n l density
(g/cm a) Each strand added directly t o Cs('l, c e n t r i f u g e d 20 h a t 25
I
E a c h strand treated separately w i t h 2 o~ f o r m a l d e h y d e in borate buffer for i h a t 3 7 ' ; strands then mixed, centrifuged in ('sC1 ~ 2 % formaldehyde
2
1.718
r.7o9
Each strand treated separately w i t h 1 % formaldehyde in borate buffer for 6 h a t 25<'; s t r a n d s t h e n m i x e d , centrifuged in C s C I ~ I ()/0 formaldehyde
2
1.727
~.7t{)
Strands mixed in borate buffer; mixture then treated w i t h i % formaldehyde for 6 h a t 25~; c e n t r i f u g e d in CsC1 1 {}0 fornmldchyde
2
1.729
1.718
Strands individually added directly t o ('sCI q r % formaldehyde; centrifuged for 2o h a t 25 ~
i
1.7()9
1.7o9
conditions, human satellite l, tile three guinea pig satellites, and two calf satellites also nucleate in analytical CsC1 density gradients 2,2a'24 whereas procaryotic DNA's do n o t n ' as. The experiments described in Table IV define further some properties of nucleation of mouse satellite DNA: (I)Nucleation depends upon the formation of hydrogen bonds, since two bands were obtained after incubating each strand separately with formaldehyde 26 (Exps. I and 2). (2) When the two strands were mixed in low salt (2o mM Na*) and formaldehyde then added, nucleation did not occur. Mixing the strands together in 7.9 molal CsC1 with formaldehyde present did result in nucleation. Tim mild conditions of the analytical ultracentrifugation (25 °, CsClplus 1 % formaldehyde) ensured that hydrogen bonds formed before the addition of formaldehyde were not broken. We therefore conclude that nucleation without significant rewinding occurred in the concentrated CsC1 solution rather than in the low salt buffer in wtfich the strands were mixed. This was confirmed by the finding that nucleation occurred when the individual strands were added directly to the CsC1 solution but not in Tris EDTA buffer (*o mM Na ~) (Table V). An increase in ionic strength has two opposing effects on the rate of renaturation: (1) It increases the rate by shielding interchain phosphate repulsions. This effect is rate-determining at low ionic strengths and influences the interactions of both homopolymers and natural nucleic acids ~7,28. (2) It decreases the rate of renaturation by fawMngintrastrand folding 1L29. A decrease in temperature has a similar effect. Tlfis effect is more pronounced with natural DNA's (e.g. T 7 D N A a°) than with poly (A) and poly (U)a8' at because the homopolymers show little or no intrastrand folding a2-a6 under conditions (temperatures greater than 252 pH 7 9, ionic strengths up to Lo) in which natural DNA's still undergo intrastrand folding 11'2u. 14io¢hz.~. Hiophys..~cta, 2 5 q ( t 9 7 2 ) 297 312
MOUSE SATELLITE D N A
303
TABLE V SALT DEPENDENCE
OF NUCLEATION
AND REXVINDING
OF SI~PARATED
STRANDS
The D N A c o n c e n t r a t i o n of the annealed H and L s t r a n d s in these e x p e r i m e n t s was 5 ttg/nll of each strand. The m i x t u r e s were added directly to the CsC1 solution after the indicated t r e a t m e n t s and centrifuged in the analytical ultracentrifuge. Solvent
T r i s - E D T A buffer o. 3 ~ NaC1 in T r i s - E D T A buffer i M NaCI in T r i s - E D T A buffer 7-9 inolal CsCI in T r i s - E D T A buffer
B u o y a n t d e n s i t y (g/cm 3) 25 ° , not annealed
Annealed • h at 38°
.4 n n e a l e d • h at 6 0 '
1.7o 9 1.7o 3 1.7o 7 1.7o 9
1.7o 9 1.7Ol 1.7o 5 1.7o8
1.709
1.699 1.7o2 1.703
Tile reassociation of mouse satellite DNA was examined in buffers of different ionic strengths at 25 °, 38° and 6o ° (Table V). Like other natural DNA's, and unlike poly(A) and poly(U), there was less rewinding of mouse satellite DNA at higher ionic strengths and lower temperatures. This is in accord with earlier results which indicate that separated strands of mouse satellite DNA show significant intrastrand folding under appropriate conditions 1°. No significant rewinding of natural DNA's occurs in concentrated CsCI due to extensive intrastrand folding. In conclusion, the ionic strength dependence of the reassociation of mouse satellite DNA resembles the properties of other natural DNA's rather than those of homopolymers. Denatured mouse satellite DNA nucleated in 7.9 molal CsC1 at 25 ° for 2o h banded at a density of 1.7o 9 g/cm 3, indicating that little rewinding occurred (Table VI). When the nucleated strands were dialyzed against either low-ionic strength buffers or moderate-ionic strength (0.3) buffers at 4 ° , unfolding of tile nucleated strands occurred and the DNA rewound to the extent that it banded at about 1.7oo g/cm ~ (Table VI). Nucleated bacterial DNA's dialyzed against similar low-ionic strength buffers at 4 ° also rewind, but not when dialyzed against moderate-ionic strength (o.3) buffers 11. Apparently, single strands of mouse satellite DNA are less extensively folded than single strands of bacterial DNA's at an ionic strength of o. 3. A plausible explanation for these results is that homopolymer-like regions in mouse satellite DNA do not prevent a high degree of intrastrand folding at high ionic strengths (Table II), but they do decrease intrastrand folding at moderate-ionic strengths. When the nucleated strands in IO mM Tris-HC1 p l u s o. 3 M KC1 (pH 7.9) were heated at 78° for 5 min, they rewound further and banded at 1.698 g/cm 3. Under these conditions, intrastrand folds melted out and rewinding then occurred. When the nucleated strands in 4 mM Tris-HC1 p l u s 0.5 mM E D T A (pH 7.9) were heated at 78° for 5 min both interstrand and intrastrand bonds melted and the DNA banded at the denatured density, 1.7o 9 g/cm ~. Thus,covalent cross-linking of tile DNA did not occur in these experiments. N e u r o s p o r a crassa nuclease 37-39 was used to investigate the reassociated products formed at different ionic strengths. This nuclease has a high degree of specificity for single-stranded nucleic acids, and therefore did not attack native mouse satellite DNA (Table VI). Denatured mouse satellite was either reassociated in 0.3 M NaC1 at 60 ° or nucleated, and then dialyzed against very-low-ionic strength buffers before diB i o c h i m . B i o p h y s . A c t s , 2.59 (i972) 297 3 I z
304 TABLE
D . M . KURNIT el al. VI
N . crassa NUCLEASE DIGESTION OF REASSOCIATED MOUSE SATELLITE D N A
Mouse satellite D N A ( i o / * g / m l ) was d e n a t u r e d at p H I2.5, neutralized, and reassociated under t h e c o n d i t i o n s listed above. The reassociated D N A ' s were t h e n d i a l y z e d e x h a u s t i v e l y at 4 ~' against t h e buffers indicated. After dialysis, 2-BE D N A samples were centrifuged in a n a l y t i c a l CsCl d e n s i t y gradients. Other 2-ttg s a m p l e s of D N A were i n c u b a t e d w i t h N. crassa nuclease for 3 ° inin at 38 ° before centrifugation. All nuelease digestions were in the presence of t o hiM MgC12.
Reassociation
conditions
Dialysis buffer
Density (g/cm a) after
Units as o / N . crassa nuclease
dialysis
Density (g/cm a) after digestion with N. crassa
nuclease Native I)NA
4 mY[ Tris-HC1
plus i o mM MgCI 2 (pH 7.9) 7-9 nlolal CsC1 in T r i s - E D T A buffer, 25 ° , 20 h
1.689
5
1.689
1.7Ol
5
1.693
1.699
5
1.692
1.7oo
8
i .696
4 mM Tris HCI
plus to nlM MgCI~ (pH 7.9) or
4 mM Tris-HC1 plus 0.5 nlM E D T A (pH 7-9) 0. 3 M NaCI in T r i s - E D T A buffer, 6 0 , 2l h 7.9 mo lal CsCI in Tris--EDTA buffer, 25 '~, 2o h
4 mM Tris-.HCl
plus IO InM MgCl~ (pH 7-9)
] o m M Tris HCI
plus o. 3 1~{ KCI (pH 7.9)
gesting with N. crassa nuclease. A nuclease-resistant core was obtained which formed a hypersharp band at a density of 1.693-1.692 g/cm 3 (Table VIi. The hypersharp bands we observed when the nuclease-resistant cores were banded in CsC1 density gradients apparently reflect the formation of high-molecular-weight networks 4'4°. This agrees with the finding of BRAHIC AND FRASER a~ that a nuclease-resistant core remains after rapidly renaturing mouse DNA is digested by N. crassa nuclease. The cores we observed had higher buoyant densities and molecular weights than those isolated by BRAHIC AND }?RASER89 under different incubation conditions and from a mixture of mouse main band and satellite DNA's. Unfortunately, it was not possible to investigate the double-stranded regions of mouse satellite DNA because the strands rewound during dialysis. The core of reassociated mouse satellite DNA which was resistant to N . crassa nuclease resembled that obtained after treatment with E. coli exonuclease ia. BRAHIC AND I;RASERa~ cited unpublished evidence of a primarily exonucleolytic reaction catalyzed by N. crassa nuclease with tRNA. Nucleation in the presence of polyribonucleotides
As shown previously (Table III), mouse satellite DNA, denatured in the presence of polyribonucleotides, bound ribopolymers poorly and formed only one band when centrifuged in an analytical CsC1 density gradient. Further experiments were conducted to favor polyribonueleotide binding over nucleation by adding a Io-fold excess of ribopolymer, and by adding, or by annealing, ribopolymer separately to t3iochim. I3iophys. Acta, 259 (i972) 297-312
MOUSE SATELLITE D N A
305
each D N A strand before mixing the complementary strands in CsC1 at 25 ° (Table VII). These conditions did not prevent nucleation, since one band only was always observed. With an excess of ribopolymer, some polyribonucleotide binding was evidenced by a heavier and broader, but always single, band. From these data, we conclude that at a I : I ratio of polyribonucleotide to DNA, nearly all of the polyribonucleotide bound to one strand can be displaced by the addition of the complementary strand. At higher ratios ( I O : I ) , nucleation still occurred, although some ribopolymer remained bound. In contrast, phage DNA's and a rapidly reassociating fraction from African green m o n k e y D N A show asymmetric polyribonucleotide binding by one strand in the presence of tile complementary strand s,15. TABLE V[I
NUCLEATION OF SEPARATED STRANDS IN THE PRESENCE OF POLYR1BONUCLEOTIDES Excess polyribonucleotide indicated that the molar ratio of polyribonucleotide phosphorus to DN'A phosphorus was i0 : i; otherwise, this ratio was I : i or 2 : I. The D N A concentration was i 0 / , g / n i l in annealing experiments, and 2/*g/ml in the CsC1 gradients. Except in the annealing experiments, binding reactions with the single strands were carried out at 25 ~' in concentrated CsCI.
Order o[ addztion
Conditic~zs
Buoyant density (g/cm a)
L+poly(U), then H or L + H , t h e n poly(U)
Add to CsC1
1.715
L+poly(U) + H+poly(U,G)
Add to CsC1
1.734 (broad band)
L + e x c e s s poly(U), then H
Add to CsC1
1.721 (broad band)
Add to CsCI
1.749
L + e x c e s s poly(U), then I f + e x c e s s poly(U,G)
Anneal L + p o l y ( U ) , add annealed mixture to CsC1, then add H
Annealed in o. 3 M
(a) Anneal L + e x c e s s poly(U), add to CsCI (b) Separately, anneal H + e x c e s s poly(U,G) + e x c e s s poly(G); add to (a)
Annealed in 0.3 M
NaCI, 6o ~', t h NraCl, 6o°, 5 h
1.716 1.7I 4
It is not clear whether the relative lability of polyribonucleotide-mouse satellite D N A complexes is due to better matching of the satellite H L strand complex or to the instability of such complexes as d A . rU 4t. The salient point is that strand displacement can occur if it leads to the formation of a more stable duplex. Strand displacement is essential for D N A renaturation: regions containing intrastrand folding must be melted out before hydrogen bonding occurs during the rewinding step. While the final equilibrium favors the formation of the interstrand complexes because of their greater stability, initial destabilization of the intrastrand bonds must o c c u r for t h e r e a c t i o n to proceed. T h u s , c o n d i t i o n s w h i c h i n c r e a s e t h e l a b i l i t y of h y d r o g e n b o n d s (high t e m p e r a t u r e , l o w ionic s t r e n g t h ) f a v o r i n t e r s t r a n d o v e r intrastrand bonding.
Stoichiometry o/the reaction When one mouse satellite DNA strand was reassociated with an unequal a m o u n t of t h e c o m p l e m e n t a r y s t r a n d , t h e r e a s s o c i a t e d c o m p l e x r e f l e c t e d t h e dis-
Biochim. Biophys. Acta, 259 (1972) 297-312
306
D.M. KURNIT el al.
proportion. The data in Table VIII demonstrate that increasing the proportion of one of the interacting strands gives rise to an increased proportion of that strand in the reassociated complex. Further, reaction conditions which favor unfolding of singlestranded molecules (lower ionic strengths, higher temperatures, longer annealing times) enable still more of the excess strands to enter the reassociated complex. Presumably, this is due to the unfolding of the limiting strand so that more of its sites become available for nucleation. After short annealing times, the stoichiometry in optimal reassociation conditions was the same as in concentrated CsC1 (about 2 : I). However, as indicated by its lighter buoyant density, the complex formed in optimal reassociation conditions had a different structure with greater helical content than the complex formed in CsC1. In other words, due to extensive intrastrand folding, complexes formed in CsC1 have fewer base pairs per interstrand bond. However, these complexes are stable in concentrated CsC1 at 25 ° because of the enhanced stability of base pairs under these conditions. These results are consistent with the finding that reassociated mouse satellite DNA consists of networks of molecules, in which strands are bound to two or more complementary strands at the same timO. TAI3LE
Vlll
STOICHIOMETRY OF MOUSE S A T E L L I T E D N A
REASSOCIATION
S e p a r a t e d s t r a n d s of m o u s e s a t e l l i t e D N A were r e a s s o c i a t e d at 6 o :' i n b o r a t e buffer plus 0 . 3 M N a C 1 for t h e t i m e s i n d i c a t e d , or at 2 5 ' i n T r i s - E D T A b u f f e r plus 7 . 9 m o l a l C s C I f o r 2 0 h . T h e t o t a l D N A p h o s p h o r u s c o n c e n t r a t i o n was 3 " I O - ~ M . U n d e r t h e s e conditions, t w o p e a k s w e r e a l w a y s o b s e r v e d in a n a l y t i c a l d e n s i t y gradients. T h e s e p e a k s c o r r e s p o n d to a free s i n g l e - s t r a n d e d D N A aud to a r e a s s o c i a t e d c o m p l e x of H a n d l~ strands. T h e s t o i c h i o l n e t r y was d e t e r m i n e d b y COulparing the relative areas under the peaks.
A1olar ratio o/ H strands
Reassociation conditions
Time o[ annealing (sec)
added per L strand z : i 4 : t : i 1 : 4 4.{~ : I 4.8 : 1 4.8 : I 4.8 : i 4.8 : i 4.8 : i 4.8: t 4.8 : t I : 2. 5
7.9 7.9 7-9 7.9 0. 3 0. 3 0. 3 0.3 0. 3 0. 3 0.3 0. 3 0. 3
m o l a l CsC1, m o l a l CsC1, m o l a l CsC1, n l o l a l CsC1, M ~ a C l , 60' M NaC1, 60' M NaC1, 60 M NaC1, 60 M NaCI, 6o M NaCI, 6o' M N a C 1 , 60' M NaC1, 60' 3/[ N a C 1 , 6 0 '
z5 25 25` 25 '
--o 1o 23 300 600 3600 72oo 75 6 0 0 3600
Buoyant denszty o[ complex (g/cm s) 1.712 1.714 1.7i 5 1.7o6 1.715 1.7o 3 1.7o 4 1.7o 7 1.7o8 1.7o 9 1.7io 1.711 1.695
Stoichiometry o[ complex (H strands pet" L strand) 1.8 2.2 2. 7 -~. j 2.2 2.I 2.2 2. 4 2. 7 2.8 3.0 3.4 . 0. 5
Strand reassociation and dissociation
As indicated by the dissociation constant of io -10 M for bacterial DNA • RNA hybrids 4z-44, strand displacement is an infrequent occurrrence with well-lnatched hybrid molecules under stringent conditions (66 °, Na ~ concentration less than I M). In contrast, strand displacement occurs in honlopolymer-homopolymer systenls miBiochbn. Biophys. ,4cta,
259
(1972)
297
312
MOUSE SATELLITE D N A
307
tochondrial DNA-homopolymer A and mouse satellite DNA-homopolymer systems (refs. 45, 55 and Table VII). Since mouse satellite DNA contains significant amounts of homopolymer-like regions and renatures to give mismatched hybrids ~'2, the reversibility of strand reassociation of mouse satellite DNA was investigated. The stoichiometry of the reassociation of mouse satellite DNA at 60 ° in 0.3 M NaC1 is shown in Table VII. These results show that a single strand of mouse satellite DNA cannot be saturated by annealing it with an equimolar amount of the complementary strand. Therefore, these experiments were performed with an excess of H strands in order to ensure saturation. The H strand was used in excess because free H strand bands in CsC1 at a density (1.728 g/cm a) which is more readily distinguished from the reassociated hybrid densities (i.69-i.7I g/cm a) than is the free L strand (1.699 g/cma). The experiments shown in Fig. I consisted of annealing an unlabeled mixture of separated strands of mouse satellite DNA at a ratio of 4.8 H strands per L strand. At different times during the annealing reaction, radioactive H strands were added to the mixture which was then annealed turther, so that the total annealing time was 2.5 h. The concentration of the radioactively labeled DNA was negligible compared to the concentrations of the unlabeled strands. When radioactive H strand was pre-
A
375F
t'O
sec
0.0,0
A 1 °°''
iii 30.c
0060
225
t004s
oooo I -D
0030
E
t /A/
o
/
Ioo,,
'
Ec
'"
~
1°''°~
300
I/
~0120
~
~'
It
~oo~o
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0 O06
150
I
5
10
15
20
25
30
35
FRACTIONNUMBER
40
45
I
5
10
15
20
25
30
FRACTIONNUMBER
35
40
a5
Fig. 1. L a b e l e d m o u s e s a t e l l i t e D N A was o b t a i n e d b y g r o w i n g m o u s e t i s s u e c u l t u r e cells in t h e p r e s e n c e of [ M e - a H J t h y m i d i n e for 3 daY s~a- U n l a b e l e d nlouse s a t e l l i t e D N A w a s a n n e a l e d a t 6o ° in b o r a t e buffer plus o. 3 1V[NaC1 a t a m o l a r r a t i o of 4.8 H s t r a n d s pe r L s t r a n d . A f t e r t h e a n n e a l ing t i n l e i n d i c a t e d l a b e l e d H s t r a n d s were a d d e d to t h e m i x t u r e , a n d a n n e a l i n g w a s c o n t i n u e d u n t i l t h e t o t a l a n n e a l i n g t i m e w a s 2. 5 h. The r e a c t i o n w a s s t o p p e d b y a d d i n g t h e n l i x t u r e t o c o n c e n t r a t e d CsC1 a t 25 °. The r e s u l t i n g 7.9 m o l a l CsC1 s o l u t i o n w a s t h e n c e n t r i f u g e d a t 32 ooo r e v . / m i n in a Spinco f i x e d - a n g l e Ti 65 r o t o r for 66 h a t 18 °. The t u b e s were p u n c t u r e d , a n d 0.25111l f r a c t i o n s were collected. Ash 0 nm ( C ) - O ) w a s d e t e r m i n e d w i t h a Zeiss P1Vf Q I [ s p e c t r o p h o t o m eter. R a d i o a c t i v i t y p r e c i p i t a t e d b y 5 % t r i e h l o r o a c e t i c acid ( Q - Q ) w a s d e t e r m i n e d on o.2inl a l i q u o t s from each fraction.
Biochim. Biophys. Acta, 259 (1972) 297-312
308
D.M.
K U R N I T et al.
sent from t h e beginning of t h e a n n e a l i n g reaction, it h y b r i d i z e d with L s t r a n d s to tile same e x t e n t as t h e u n l a b e l e d H s t r a n d s (Fig. I a ) . L a b e l e d H s t r a n d s a d d e d l a t e r d u r i n g the annealing r e a c t i o n showed progressively less reaction with L s t r a n d s (Figs. I b - I d ) . Therefore, at some p o i n t in their reassociation process, s t r a n d s of mouse satellite D N A become s t a b l y paired. I t is possible to e s t i m a t e a second-order r a t e c o n s t a n t (k~) for the reaction bet w e e n one L s t r a n d a n d one H s t r a n d from t h e d a t a in T a b l e V I I I . A f t e r a fraction of t h e reassociation process, N, has been c o m p l e t e d , t h e effective c o n c e n t r a t i o n s of H a n d L s t r a n d s are (PT/5.8) • ( 4 . 8 - - S N ) a n d (PT/5.8) • ( I - - N ) respectively, where PT is t h e t o t a l D N A p h o s p h o r u s c o n c e n t r a t i o n a n d S is t h e n u m b e r of H s t r a n d s p r e s e n t in t h e r e a s s o c i a t e d c o m p l e x per L s t r a n d at t h e t i m e indicated. F o r a secondo r d e r reaction, d
EPr
dt 51s'('--X)
]
PT.(4.8
=*~'5.s
SN)PT
.5-- (~--x)
(i)
where t is t h e annealing t i m e before the r a d i o a c t i v e H s t r a n d s were added. E a c h value of N was corrected for the change in S which occurs with increased annealing time. Eqn. I can be i n t e g r a t e d to yield t h e following relation: l . 4 .8 - - / (
( In ] 4~ " s - S N. I
,
.
-,u(4.s))=k, l~' . t . . 5.
(2)
F r o m T a b l e I X , t h e average value of k 2 is 6 • IO ~ 1 • mole ~ • sec-L The l i t e r a t u r e value of k 2 for mouse satellite D N A reassociated in o . i 8 M N a -~ is 1. 5 • i o ~ 1 • mole 1. sec - t (ref. 46), which corresponds to a k 2 of 3.5 " IOa 1 • mole - t • sec -1 in 0. 3 M N a + (ref. 47)- The m o s t likely e x p l a n a t i o n for t h e low k 2 value of Table I X is t h a t t h e f u r t h e r b i n d i n g of excess H s t r a n d s to the reassociated c o m p l e x is e n c u m b e r e d b y H s t r a n d s a l r e a d y in t h e complex. T h e kinetic studies p r e s e n t e d here i n d i c a t e t h a t a s t r a n d of mouse satellite D N A s t a b l y b o u n d to its c o m p l e m e n t will n o t be displaced b y a c o m p e t i n g D N A TAI3LE IX DETERMINATION
OF THE
RATE
OF RFASSOCIATION
FROM STRAND
COMPETITION
DATA
A n n e a l i n g of m o u s e s a t e l l i t e D N A w a s p e r f o r m e d as d e s c r i b e d in t h e l e g e n d t o Fig. i. I n o r d e r t o c a l c u l a t e N , i t w a s a s s u m e d t h a t N = o a t t = o, a n d N - - l a t t ~ 2. 5 h . ( I - - N ) w a s d e t e r m i n e d for e a c h v a l u e of t b y d i v i d i n g t h e f r a c t i o n of c o u n t s i n t h e r e a s s o c i a t e d c o m p l e x a f t e r t h i s a n n e a l i n g t i m e b y t h e s a m e f r a c t i o n w h e n t ~ o. T h i s v a l u e of N w a s t h e n c o r r e c t e d for the change in S which occurs with increased annealing time (Table Vlll). The total DNA phosp h o r u s c o n c e n t r a t i o n in t h e s e e x p e r i m e n t s v a r i e d f r o m 3 " i o 5 t o 3 . 5 ' lO-5 M~. Time o/annealing be/ore a d d i t i o n o/ labeled H s t r a n d (see) (t)
0
3° 60 3° o 36oo
F r a c t i o n o/ L strands u n a v a i l a b l e / o r reaction with labeled H s t r a n d (N)
Second-order rate constant (k2) d e t e r m i n e d [rom Eqn. 2 (1 • mole -1 • sec -1 • lO -2 )
0 . 0 0
- -
0.36 0.54 °'91 °'98
6
B i o c h i m . B i o p h y s . A c t a , 259 (1972) 297--312
6 4 7
MOUSE SATELLITE DNA
309
strand. The kinetics of the strand competitionlreaction are second order. The predominant reaction at equilibrium is HsL ~ H s _ I L + H , where S is the number of H strands per L strand at equilibrium. The dissociation constant, KD, is defined as follows:
KI~
[Hs tL~ [HI [HsL]
(3)
Using the relations
PT [Hs_~L][HsL] I--NNand [H] =5.8 " (4"8--SN) and substituting the appropriate values of S and N determined at t = 3600 sec, the calculated value for K n is about IO 7 M. Since the reaction has not reached equilibrium at this time, this is an estimate of the m a x i m u m limit of KD. In addition, preliminary results using this strand competition method indicate that strands nucleated in concentrated CsC1 for 6 h are stably bound.
Mouse satellite DNA--how simple? Although the nucleotide organization of mouse satellite DNA is more complicated than that of homopolymers, it is simpler than that of other natural DNA's. Mouse satellite DNA exhibits some physical properties resembling those of natural DNA's and other properties resembling those of homopolymers. It resembles homopolymers in that: (I) A significant portion of the DNA is composed of the pyrimidine tract TTTTTC and related variations 5. Polyribonucleotide binding studies confirm the presence of T-rich sequences on the H strand, and, in addition, indicate the presence of (A,C)-rich (or C-rich) sequences on the H strand. (2) The base compositions of the complementary strands are unequal, as illustrated by the large buoyant density differences of the two strands in alkaline CsC12,~. (3) The buoyant density and melting temperature of the duplex molecule are not equal to those predicted from its base composition 1-3. (4) Synthetic polyribonucleotides (and possibly natural RNA's) bound to one DNA strand m a y be displaced by the addition of the complementary DNA strand. On the other hand, mouse satellite DNA has several properties which resemble those of other natural DNA's. The base composition and melting temperature show that it is not merely a polymer of the type dA. dT. The buoyant densities of the single strands (Table I) agree with the densities calculated from the base composition of each strand by an empirical equation which relates the base composition and buoyant densities of single-stranded procaryotic DNA's. The reassociation of mouse satellite DNA is inhibited at high ionic strengths and low temperatures because of intrastrand folding, and gives rise to an essentially irreversible product.
DISCUSSION Reassociation of DNA molecules in 7.9 molal CsC1 at 25 ° involves nucleation without significant rewinding. It can be estimated n from the buoyant density of the nucleated strands that the extent of rewinding is less than 5 %- Thus, reassociation in concentrated CsC1 at 25 ° is determined by the repetition frequency of short nucleotide sequences. Mouse satellite DNA, human satellite I, the three guinea pig satellites Biochim. Biophys. Acta, 2.59 (1972) 297-312
310
D . M . KURN|T
el al.
and two calf satellites all nucleate in analytical CsC1 density gradients(7.()molal CsC1, 25 °, 2o h) TM. This contrasts with h u m a n main band D N A (unpublished observations) and procaryotic D N A ' s which do not nucleate under these conditions. However, procaryotic D N A ' s will nucleate in concentrated CsC1 at 25" over a period of days 11''~5, as will lmman main band DNA at high concentrations (unpublished observations). Mouse satellite and guinea pig satellite I I)NA apparently contain a high degree of r e d u n d a n c y due to the multiplication and subsequent divergence of short (6 13 nucleotides) sequences 5. The structure proposed for these D N A ' s is consistent with their rapid nucleation rate in CsC1 at 2 5 ' and with the observed polyribonucleotide interactions. Despite its rapid nucleation in CsC1 at e5 °, guinea pig satellite I D N A in o.I8 M Na ~ at 6o ~"gives a broad, shallow Cot curve consistent with a repeated sequence of about IO5 nucleotide pairs ~,24. In contrast, a series of bacteriophages from Bacillus stearotherm@hihts and B. subtilis with genome sizes ranging from 4" ~o4 to 2 • io ~ nucleotide pairs shows ahnost no nucleation in 7.q molal Cs('l after 2o h 4~ ~0. The rate of nucleation in CsC1 is at least a qualitative measure of complexity ("the n u m b e r of base pairs in non-repeating sequences per virus or cell for the given D N A ''1~) for procaryotic D N A ' s since Hemophilus in/lue~za DNA, which is five times larger than T 4 DNA, nucleates at about 4o (.!i~of the rate of the phage DNA TM2a. Presumably, guinea pig satellite I D N A strands nucleate more frequently than bacteriophage D N A ' s in CsC1 at z5", but fewer of the nucleations remain stable under the more restrictive conditions (6(¢, o.I8 M Na~). The nucleation of short nucleotide sequences of procaryotic D N A ' s leads to binding of proper D N A complements 19. As a result, the complementary regions adiacent to the nucleation sites are brought into apposition and provide the homology required for stability under more stringent conditions. In contrast, the nucleation of animal satellite D N A ' s inw)lves the pairing of short nucleotide sequences which are not on proper c o m p l e m e n t a r y strands; regions adiacent to the nucleation site need not be complementary. Therefore, these nucleations are not necessarily stable under more stringent conditions. In other words, the " c o m p l e x i t y " measured at z5 '~ in concentrated CsC1 and t h a t measured at 6o" in o.3 M NaC1 are not the salne: the former reflects the repetition frequency of much shorter nucleotide sequences than does the latter. The complexity of procaryotic D N A ' s measured at 6o" in o.I8 M Na ~ correlates with the complexity measured at 25 '~ in concentrated Cs('l H,~. This correlation does not apply in the case of mouse satellite and guinea pig satellite l DNA's. Unless the type of sequence homology under investigation is clearly defined, the term " c o m p l e x i t y " is ambiguous when applied to these animal satellite DNA's. These D N A ' s apparently consist of reFeated nuclcotide sequences shorter than indicated from the Cot value and which are heterogeneous and/or interspersed between non-repeated sequences ",5,24. As the degree of mismatching increases, the apparent sequence length calculated from reassociation kinetics at restrictive conditions also increases. Accordingly, guinea pig satellite I DNA, which shows more mismat ching after reassociation than mouse satellite a,a.'4, has an apparent sequence length about one hundred times greater than t h a t of mouse satellite DNA under restrictive conditions 2~. Recently, SOUTHERNs~ and SWTON a~t) McCALLUM5'2 have also concluded t h a t mismatching has a significant effect on the rate of reassociation of nucleic acids. Biochim. t4iophys..4cta, 239 (1072) 297 312
MOUSE SATELLITE D N A
311
ACKNOWLEDGEMENTS
We thank Mrs. A. Tobia for her gift of labeled mouse satellite DNA and Dr. J. Hurwitz for his gift of N . c r a s s a nuclease. These studies were supported by grants from the National Science Foundation, the American Cancer Society, and the National Institutes of Health (AI 4153). D. M. K. is a Medical Scientist Trainee supported by U.S. Public Health Service grant No. 5T 5 GM 1674. J.J.M. and C.L.S. are recipients of National Institutes of Health Career Development Awards. REFERENCES I~. E. ]3OND, W. G. FLAMM, H. E. ]3URR AND S. ]3. ~3OND, J . Mol. Biol., 27 (1967) 289. G. CORNEO, E. GINELLI, C. SOAVE AND G. BERNARDI, Biochemistry, 7 (1968) 4373. (-'. L. SCHILDKRAUT AND J. J. MAIO, Biochim. Biophys. Acta, 161 (1968) 76. M. \VARING AND R. J. ]3RITTEN, Science, 154 (1966) 79 I. E. M. SOUTHERN, Nature, 227 (197 o) 794. J. MARMUR, J . Mol. Biol., 3 (1961) 208. \.V.G. FLAMM, M. MCCALLUM ANn P. M. [[:3. WALKER, Proc. Natl. Xead. Sci. U.S., 57 (I967) 1729. 8 J. J. MAIO, .]. Mol. Biol., 56 (1971 ) 5799 S. |~.IVA, 1. BARRAI, L. CAVALLI SFORZA AND A. FALASCHI, J. Mol. Biol., 45 (1969) 367. l O W . G. FLAMM, P. M. ]3. WALKER AND M. MCCALLUM, J. Mol. Biol., 4 ° (1969) 423 . 11 }¢.. H. ROWND, P h . D . Thesis, Harvard University, C a m b r i d g e , Mass., U.S.A., 1963. i2 C. L. SCHILDKRAUT AND J. J. MAIO, J. ]Viol. Biol., 46 (1969) 305 . 13 H. KUBINSKI, Z. OPARA--KUBINSKA AND W. SZYI3ALSKI, J. Mol. Biol., 20 (1966) 313 . 14 IC BURTON AND G. ]3. PETERSEN, Biochem. J., 75 (196o) 17. 15 \ v . SZYBALSKI, H. KUBINSKI AND P. SHELDRICK, Cold Spring Harbor Syrup. Quant. Biol., 3 t (1966) I23. i6 J. HAREL, N. HANANIA, H. TAPIERO AND L. HAREL, Biochem. Biophys. Res. Commun., 33 (1968) 696. 17 J. I'V[ARMUR, |{. ]{OXVND AND C. L. SCHILDKRAUT, Prog. Nucleic dcid Res. Mol. Biol., 1 (1963) 231. 18 J. G. \VETML'R AND N. DAVIDSON, J. ~lol. Biol., 31 (1968) 349' 19 C. A. THOMAS, JR., Prog. Nucleic dcid Res. IVIol. Biol., 5 (1966) 315 . 2o S. K. NIYOGI, J. Biol. Chem., 244 (1969) 1576. 21 P. M. t3. WALKER, Prog. Nucleic dcid Res. Mol. Biol., 9 (1969) 3Ol. 22 R. \Vu, J. 2~lol. Biol., 51 (197 ° ) 5Ol. 23 G. CORNEO, E. GINELLI AND E. POLLI, f . Mol. Biol., 48 (197o) 319. 24 G. CORNEO, E. GINELLI AND E. POLLI, Biochemistry, 9 (197 o) t565 . 25 J. A. SUBIRANA AND I9. DOTY, Biopolymers, 4 (1966) 17126 L. GROSSMAN, S. S. LEVlNE AND V~T. S. ALLISON, J. Mol. Biol., 3 (1961) 47. 27 J. MARMUR AND D. LANE, Proc. Natl. ,4cad. Sci. U.S., 46 (196o) 45328 P. D. R o s s AND J. M. STURTEV.A_NT, J. ,4m. Chem. Soc., 84 (1962) 45o3 . 29 F. VV. STUDIER, f . ;'VIol. Biol., 41 (1969) 199. 3 ° F. W. STUDIER, J. 3Iol. Biol., 41 (1969) 189. 31 [{. D. BLAKE AND J. ]{. FRESCO, J. IVIol. Biol., 19 (1966) 145. 32 M. LENG AND G. FELSENFELD, J. ~/[ol. Biol., 15 (1966) 45533 J. ]3RAHMS, A. M'. MICHELSON AND lX. E. VAN HOLDE, J. 2V[ol. Biol., 15 (1966) 467 . 34 J. c. TrlRIERR AND ~f. LENG, Biochim. Biophys. Acta, i82 (1969) 57535 I). ]3. MILLAR AND M. 5~ACKENZlE, Biochim. Biophys. Acta, 204 (197 o) 82. 36 L. D. INNERS AND G. FELSENFELD, J. Mol. Biol., 5 ° (197 o) 373. 37 E. Z. RABIN AND M. J. FRASER, Can..]. Bioehem., 48 (197 o) 389. 38 S. LINN AND I. R. LEHMAN, J. Biol. Chem., 240 (1965) 1287. 39 M. BRArtlC AND M. J. FRASER, Biochim. Biophys. dcta, 240 (1971) 23. 4 ° 1. ]3. DAWlD AND D. R. W'OLSTENHOLME, Biophys. J ., 8 (1968) 65. 41 5.][. CHAMBERLIN, Fed. Proc., 24 (1965) 1446. 42 R. LAVALL~ AND G. DE HAUWER, J. Mol. Biol., 37 (1968) 269. 43 J- o. ]31SHOP, Biochem. J., 116 (197 ° ) 223 . 44 D. E. KENNELL, Prog. Nucleic dcid Res. Wlol. Biol., i i (1971) 259. 45 M. J. CHAMBERLIN AND D. L. PATTERSON, J. Mol. Biol., 12 (1965) 41o. 46 R. J. ]3RITTEN AND D. E. KOHNE, Science, 161 (1968) 529. t 2 3 4 5 6 7
Biochim. Biophys. Acta, 259 (1972) 297 312
312
D . M . KURNIT et al.
47 R. J. ~[~R1TTEN AND J. SMITH, Carnegie Institution Yearbook, 68 (1969) 378. 48 S. CORDES, H. T. EPSTEIN AND J. MARMUR, Nature, 19i ( i 9 6 i ) lO97. 49 J. MARMUR AND S. CORDES, in H. J. VOGEL, V. ~BRYSEN AND J. o . LAMPEN, ln/ormational Macromolecules, A c a d e m i c Press, N e w York, 1963, p. 79. 50 G. F. SAUNDERS AND L. L. CAMPBELL, Biochemistry, 4 (1965) 2836. 51 E. M. SOUTHERN, Nature New Biol., 232 (1971) 82. 52 W. D. SUTTON AND ~ . MCCALLUM, Nature New Biol., 232 (i97 t) 83. 53 W. G. FLAMM, P. M. B. WALKER AND M. MCCALLUM, J. Mol. Biol., 42 (1969) 441. 54 A. M. TOBIA, C. L. SCHILDKRAUT AND J. J. MAIO, Bioehim. Biophys. Acta, 246 (1971) 258. 55 P- BORST AND (9. J. C. M. ]~UTTENBERG, Biochim. Biophys..4eta, 19o (i969) 391.
Biochim. Biophys. Acta, 259 (1972) 29(J 312
297
BBA 97136
S I N G L E - S T R A N D I N T E R A C T I O N S OF MOUSE S A T E L L I T E DNA D A V I D M. K I ; R N I T , C A R L L. S C H I L D I ( R A U T AND J O S E P H J. ~.rAIO
Division o[ Biologica/ .q'ciences (Cell Biology), .4lbert Einstein Cv/lege o/ 3Iedicinc, t3ronx, N.3". Lo46~ (U.S..-I.) (Received A u g u s t 24th, 1971)
SUMMARY
Interactions of the complementary strands of mouse satellite DNA were investigated in the presence of polyribonucleotides, at high ionic strengths, and at different molar ratios of the two strands. Polyribonucleotide binding demonstrated the presence of T-rich and (A,C)-rich or C-rich sequences on the heavy strand. Ribopolymers bound to one strand of mouse satellite DNA were displaced by addition of the complementary DNA strand. However, the formation of mouse satellite DNA • DNA hybrids is essentially irreversible. High ionic strengths and formaldehyde were used to investigate the two steps, nucleation and rewinding, which comprise the reassociation of mouse satellite DNA. Compared with procaryotic DNA's, the complementary strands of mouse satellite DNA nucleate rapidly. The rewinding of nucleated complementary strands of mouse satellite DNA is inhibited at high ionic strengths and low temperatures. The effects of rapid nucleation of mismatched strands on the reassociation of animal satellite DNA's are discussed.
INTRODUCTION
Unlike most naturally-occurring DNA's, the buoyant density and melting temperature of mouse satellite DNA do not correspond to the values predicted from its base composition 1-3. In addition, the separated strands reassociate rapidly 4 to form a mismatched hybrid 1,2. These properties are due to the nucleotide organization of mouse satellite DNA, which apparently contains repeats of (and variations on) a homopolymer-like sequence, TTTTTC ~. This report describes the interactions of the complementary strands of mouse satellite DNA in buffers of various ionic strengths in the presence of polyribonucleotides, and at different ratios of the two strands. The results demonstrate a correlation between the nucleotide organization of mouse satellite DNA and its reassociation properties.
METHODS AND MATERIALS
Nuclear DNA from a mouse neuroblastoma tissue-culture line with a molecular weight of approx, lO 7 was obtained by a modification of MARMUR'S3'6 method. Mouse satellite DNA was isolated by preparative density-gradient centrifugation in Biochim. Biophys. ~4cta, 259 (I972) 297-312
1). M. KURNIT t'[ al.
298
A g ÷ Cs2SO 4 (ref. 2} d i a l y z e d a g a i n s t 2 M NaC1 in T r i s - E D T A b u f f e r (IO m M Tris HC1 plus I m M E D T A (pH 8.4) ), a n d t h e n d i a l y z e d a g a i n s t T r i s - E D T A buffer. T h e c o m p l e n l e n t a r y h e a v y (H) a n d ligllt (L) s t r a n d s of m o u s e s a t e l l i t e D N A were sep a r a t e d b y c e n t r i f u g a t i o n in p r e p a r a t i v e a l k a l i n e Cs('l (pH 12.5) density" g r a d i e n t s 7. T h e s e p a r a t e d s t r a n d s w e r e t h e n d i a l y z e d a g a i n s t T r i s - E D T A buffer. TAI3LE 1 BUOYANT
DENSITIES
OF ANIMAL
SATELLITE
[)NA's
Buoyant density and base composition data for the duplex satellite DNA's and their complementary strands were obtained from the sources listed under Re/erence. Buoyant densities of the duplex molecules in CsC1 were calculated from their base compositions according to the equation: f, ~- o.o9S ((; t C)+ ~.659 {~) where p is the DNA buoyant density in g/cm a and (G ! C) is the average mole fraction of Rimnine and cytosine ~2. ]3uoyant densities of the single-stranded molecules in CsC1 at pH 8. 4 were calculated from their base compositions according to the equation: p - - 1.62(~69A-}-1.758o4G~ ~.768o9 C ~ 1.7424IT {2) where p is the DNA buoyant density in g/cm a and A, G, C and T are the average mole fractious of each of the four bases 9. The density of E. coli native DNA was taken as 1.71o g/cm 3 in all tmoyant density determinations.
Satellite 1)XA
Re/erence
Mouse satellite
2,3,7; Table I1 this paper
Duplex L strand H strand Guinea pig satellite I Duplex I. strand H strand Human satellite [ Duplex 1. strand H strand
Observed buoya~z! density (g/cm 3)
Buoyant densitiy (g/cma)/rom Eq~2. r (double stranded DN.4)
1.689 i .699 i .728
1.695
1.7o 5 1.7o6 .754
i .608
i .686 1.694 1.712
~.685
Buoyant dealsity (g/cma)#om Eqn. 2 (singh,stranded DNA )
[ .608 t.727
53 i. 709 t.724
23, 12 ---
i .696 1.712
A n a l y t i c a l d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n in a S p i n c o M o d e l E u l t r a c e n t r i f u g e w a s as d e s c r i b e d p r e v i o u s l y a. I n n e u t r a l CsC1, D N A f r o m t h e Bacillus subtilis b a c t e r i o p h a g e 6C (density, 1.742 g / c i n a) a n d p o l y E d ( A - T ) -d(A-T)I~ (density, 1.679 g / c m a) w e r e u s e d as s t a n d a r d s for d e n s i t y d e t e r m i n a t i o n . T h e s e v a l u e s c o r r e s p o n d to a d e n s i t y of 1.71o g / e m 3 for Escherichia coli D N A . I n a l k a l i n e CsCl (pH I2.5), t h e h e a v y s t r a n d of t h e B. subtilis b a c t e r i o p h a g e 6C D N A was t a k e n to be 1.8Ol g/cin "~, c o r r e s p o n d i n g to a v a l u e of 1.772 g / c m a for E. coli D N A c e n t r i f u g e d u n d e r similar c o n d i t i o n s . F o r e e n t r i f u g a t i o n in t h e p r e s e n c e of f o r m a l d e h y d e , t h e D N A was d i a l y z e d a g a i n s t b o r a t e b u f f e r (5 inM N a 2 B 4 0 7 p l u s 5 m M Na2SO 4 (pH 8.8)). T h e a n a l y t i c a l u l t r a c e n t r i f u g a t i o n was in CsC1 d i s s o l v e d in b o r a t e buffer. T h e f o r m a l d e h y d e was d i l u t e d f r o m a 4 ° °~b s t o c k s o l u t i o n of f o r m a l i n (Merck).
Biochim. Biophys. Acta, 259 (i972) 297-312
MOUSE SATELLITE DNA
299
Polyribonucleotide binding The preparation and calibration of the stock solutions of polyribonucleotides were described in an earlier paper s. Polyribonucleotides were added to DNA in T r i s - E D T A buffer to the desired ratio of polyribonucleotide phosphorus to DNA phosphorus in a total volume of IiO/~1. Unless otherwise stated, this ratio was I : I or 2 : 1 . Initially, experiments were performed by heating tile native or singlestranded DNA samples in T r i s - E D T A buffer at 9 °o for 5 rain in the presence of polyribonucleotides. It was later found that heating at 9 °o was not necessary for binding to the single strands. The same results were obtained when tile polyribonucleotides were added directly to the DNA in Tris EDTA buffer or in CsC1 at 25 °.
RESULTS AND INTERPRETATION
Properties o/the isolated single strands The densities in CsC1 (pH 8.4) of the complementary strands of mouse satellite DNA were 1.728 g/cm a and 1.699 g/em a, in agreement with previously published values (ref. 2 and Table II). The bands formed in CsC1 were sharp and symmetrical, with no shoulders. The buoyant densities of the single strands coincided with the densities calculated from the base composition of each strand by means of an empirical equation which relates the base composition and buoyant densities of singlestranded DNA's (ref. 9 and Table I). Interestingly, this equation applies to all of tile single strands of the satellite DNA's listed in Table I with the exception of the H strand of guinea pig satellite I DNA. TABLE BUOYANT
II DENSITIES
OF MOUSE SATELLITE
DUPLEX
AND SINGLE-STRANDED
1)*~N~A
Expt. No.
Sample
Duplex
H strand
L strand
I 2
D u p l e x i n CsC1 ( p H 8.4) D u p l e x in T r i s E D T A b u f f e r , 5 / * g / m l , h e a t e d a t 9 °o f o r 5 rain and fast cooled D u p l e x in CsC1 ( p H 12.5) S e p a r a t e d s t r a n d i n CsCl ( p H 8.4) S e p a r a t e d s t r a n d in T r i s E D T A b u f f e r , 5 ~¢g/ml, h e a t e d at 9 °° for 5 min S e p a r a t e d s t r a n d in o. 3 M N a C I , 5 t l g / m l , heated at 60 ° for i h
1.689
--
--
1.71o --
-1.767
-1.742
---
1.728
1.699
--
1.728
1.699
--
1.729
1.699
3 4 5
6
As measured by hydroxyapatite chromatography and absorbance, a significant amount of intrastrand folding occurs when either H or L strands are self-annealed 1°. However, changes in intrastrand folding introduced by denaturing or annealing single strands before density gradient centrifugation did not affect the final buoyant densities (Table II). The high ionic strength of the 7.9 molal CsC1 solution enhances the stability of hydrogen bonds and increases intrastrand folding n. It is probable Biochim. Biophys. Acta, 2 5 9 ( 1 9 7 2 ) 2 9 7 - 3 1 2
300
D.M. KURNIT e[ al.
t h a t the high degree of i n t r a s t r a n d folding of s i n g l e - s t r a n d e d D N A ' s in c o n c e n t r a t e d CsC1 solutions m a s k s pre-existing differences in i n t r a s t r a n d h y d r o g e n bonding. F r a c t i o n s from the light a n d h e a v y sides of each single s t r a n d isolated in the p r e p a r a t i v e alkaline CsC1 d e n s i t y g r a d i e n t were rerun in a n a l y t i c a l alkaline CsC1 d e n s i t y gradients. H s t r a n d fractions b a n d e d from L768 to L765 g/cma; L s t r a n d fractions r a n g e d from 1.743 to 1.741 g/cm :3. The reassociation characteristics of these fractions confirmed t h a t c o n t a n f i n a t i n g m a i n b a n d D N A was not present. Thus, a l t h o u g h mouse satellite D N A is more homogeneous t h a n m a i n b a n d D N A t2, d e n s i t y h e t e r o g e n e i t y was detectable. H e t e r o g e n e i t y in the basic nucleotide sequence of mouse satellite D N A has been o b s e r v e d p r e v i o u s l y 5. l~olvribonucleotide binding H e a t - d e n a t u r e d m a i n b a n d D N A b o u n d p o l y ( U ) , poly(G), a n d p o l y ( U , G), a n d showed the b u o y a n t d e n s i t y increase which is c h a r a c t e r i s t i c of a n i m a l D N A ' s ~3 (Table In). The single b r o a d p e a k o b t a i n e d was due p r e s m n a b l y to d e n s i t y heterog e n e i t y a n d to b i n d i n g of a p p r o x i m a t e l y equal a m o u n t s of p o l y r i b o n u c l e o t i d e s b y b o t h s t r a n d s of t h e D N A molecules. W h e n d u p l e x satellite D N A was r e a c t e d w i t h t h e p o l y r i b o n u c l e o t i d e s u n d e r similar conditions, n m c h less b i n d i n g was d e t e c t e d . However, the D N A b a n d e d at a b o u t the d e n a t u r e d density. In contrast, the s e p a r a t e d
TAI~LE II[ POLYRIBONIJCL],;OT1DFBINDINGTO rqOUSl"I)~N~.\ Sample
Polyribom~cleolide (s) added
Huoyant dcnsilv (g/cm 3)
Mouse main band, 25' MZouse nlain band, 9 0 5 lnill
]N*Olle
1.7oo
.'NTOile Poly(IT) Poly(G) Poly(l',G)
~.715 r.729 1.726 1.756
None
1.689
~-N'OIIe Poly(U) Poly(G) Poly(IT,G}
1.71o 1.7I 4 1.71o 1.715
~Olle Poly(U) Poly(G) Poly(U,G)
.699 1.788 1.698 1.7o8
None Poly(U) Poly(G) Poly(U,Z~) Poly(t',G) ] Poly(G)
1.728 1.727 1.742 1.779 1.773
NIouse satellite, duplex, 2 5 5'[ouse satellite, duplex, 9o', 5 rain
Mouse satellite, separated L strand
Mouse satellite, separated H strand
Biochim. Iliophys..-tcta, 259 (i972 ) 297-312
Huoyanl density increment over denatured de~lsity (mg/cm~)
o 14
ii 4 r
89 o 9
o
14 5I 45
MOUSE SATELLITE
DNA
3Ol
strands of mouse satellite DNA bound polyribonucleotides in a highly specific and asymmetric pattern. Poly(U) bound only to the L strand, and resulted in a buoyant density increment of 89 mg/cm 3 (Table I I I ) . This was the largest density increment we observed with this technique in any animal or microbial DNA. The asymmetric pattern was further demonstrated by the selective binding of poly(U,G) and poly(G) to the H strand. The binding sites for poly(U, G) and poly(G) apparently coincide, since the addition of both ribopolymers did not have an additive effect on the buoyant density increment of the H strand. Complexes between the separated DNA strands and polyribonucleotides formed readily at 25 ° in 7-9 molal CsC1. Poly(A) and poly (A,C) did not bind, perhaps because of specific conformational effects. We conclude that mouse satellite DNA H strand contains extensive regions of T-rich sequences as evidenced by the large amount of poly(U) binding by the L strand. This is consistent with pyrimidine tract data which suggest that TTTTTC is included in the basic sequence s. The presence of poly(U,G) and poly(G) binding sites on the H strand suggests that there is another repeated sequence, or that (A,C)-rich (or C-rich) clusters fill out the remainder of the basic sequence which is postulated to be 8-13 nucleotides long 5. Since (A,C)-rich sequences are disrupted by diphenylamine-formic acid hydrolysis 14, this sequence would not be detected by pyrimidine tract analysis. Main band DNA contains ribopolymer binding sites as does satellite DNA (Table I I I ) ; it is possible that these sequences account for the limited binding of satellite DNA to main band DNA 1°. Asymmetric polyribonucleotide binding has been observed in bacterial and bacteriophage DNA's 1~,15, and in a rapidly reassociating fraction of African green monkey DNA s. In bacteriophages, the presence of pyrimidine-rich sequences on one strand has been associated with preferential transcription of that strand 15. By this criterion, only the H strand of mouse satellite DNA would be transcribed. However it is not clear whether mouse satellite DNA is transcribed in vivo 1°'16. The e//ects o/ ionic strength on reassociation Renaturation of nucleic acids appears to be a two-step process 17,18. The first step, nucleation, is a bimolecular reaction involving the collision and complementary pairing of a limited number of base pairs. The smallest number of such base pairs forming a duplex region stable enough to permit further helical growth is termed the stable minimum length 19. The second-order rate constant of the nucleation reaction depends directly upon the number of complementary nucleotides on the interacting strands long enough to function as a stable minimum length. Since the stable minim u m length need be only about 12-3o nucleotides long 2°-22, nucleation is a measure of the homology of short nucleotide sequences on the interacting molecules. The second step, rewinding, is a unimolecular reaction in which helical growth follows successful nucleation. This reaction also varies with the base sequence homology of the interacting DNA's, but in a more complex way than the nucleation reaction. When mouse satellite DNA H and L strands were mixed and centrifuged in an analytical CsC1 density gradient (7.9 molal CsC1, 20 h, 25°), one band at a density of 1.709 g/cm 3 was observed (Table IV). The density of this band was 0.02 g/cm 3 heavier than the native density of 1.689 g/cm ~. This density increase is characteristic of denatured DNA. The presence of only one band at the denatured DNA density indicated that nucleation had occurred without significant rewinding. Under these Biochim. Biophys..4eta, 259 (1972) 297-312
302 TABLE
1). M. K U R N I T e~ ( d . IV
FORMALDEHYDE
Expt. No.
REACTION
~,VITtt S E P A R A T E D
STRANDS OF MOUSE SA.TELLITE ])N ,\
1 nc~tbation conditions
Number o/ I)N.4 bands observed
1~uoya n l density
(g/cm a) Each strand added directly t o Cs('l, c e n t r i f u g e d 20 h a t 25
I
E a c h strand treated separately w i t h 2 o~ f o r m a l d e h y d e in borate buffer for i h a t 3 7 ' ; strands then mixed, centrifuged in ('sC1 ~ 2 % formaldehyde
2
1.718
r.7o9
Each strand treated separately w i t h 1 % formaldehyde in borate buffer for 6 h a t 25<'; s t r a n d s t h e n m i x e d , centrifuged in C s C I ~ I ()/0 formaldehyde
2
1.727
~.7t{)
Strands mixed in borate buffer; mixture then treated w i t h i % formaldehyde for 6 h a t 25~; c e n t r i f u g e d in CsC1 1 {}0 fornmldchyde
2
1.729
1.718
Strands individually added directly t o ('sCI q r % formaldehyde; centrifuged for 2o h a t 25 ~
i
1.7()9
1.7o9
conditions, human satellite l, tile three guinea pig satellites, and two calf satellites also nucleate in analytical CsC1 density gradients 2,2a'24 whereas procaryotic DNA's do n o t n ' as. The experiments described in Table IV define further some properties of nucleation of mouse satellite DNA: (I)Nucleation depends upon the formation of hydrogen bonds, since two bands were obtained after incubating each strand separately with formaldehyde 26 (Exps. I and 2). (2) When the two strands were mixed in low salt (2o mM Na*) and formaldehyde then added, nucleation did not occur. Mixing the strands together in 7.9 molal CsC1 with formaldehyde present did result in nucleation. Tim mild conditions of the analytical ultracentrifugation (25 °, CsClplus 1 % formaldehyde) ensured that hydrogen bonds formed before the addition of formaldehyde were not broken. We therefore conclude that nucleation without significant rewinding occurred in the concentrated CsC1 solution rather than in the low salt buffer in wtfich the strands were mixed. This was confirmed by the finding that nucleation occurred when the individual strands were added directly to the CsC1 solution but not in Tris EDTA buffer (*o mM Na ~) (Table V). An increase in ionic strength has two opposing effects on the rate of renaturation: (1) It increases the rate by shielding interchain phosphate repulsions. This effect is rate-determining at low ionic strengths and influences the interactions of both homopolymers and natural nucleic acids ~7,28. (2) It decreases the rate of renaturation by fawMngintrastrand folding 1L29. A decrease in temperature has a similar effect. Tlfis effect is more pronounced with natural DNA's (e.g. T 7 D N A a°) than with poly (A) and poly (U)a8' at because the homopolymers show little or no intrastrand folding a2-a6 under conditions (temperatures greater than 252 pH 7 9, ionic strengths up to Lo) in which natural DNA's still undergo intrastrand folding 11'2u. 14io¢hz.~. Hiophys..~cta, 2 5 q ( t 9 7 2 ) 297 312
MOUSE SATELLITE D N A
303
TABLE V SALT DEPENDENCE
OF NUCLEATION
AND REXVINDING
OF SI~PARATED
STRANDS
The D N A c o n c e n t r a t i o n of the annealed H and L s t r a n d s in these e x p e r i m e n t s was 5 ttg/nll of each strand. The m i x t u r e s were added directly to the CsC1 solution after the indicated t r e a t m e n t s and centrifuged in the analytical ultracentrifuge. Solvent
T r i s - E D T A buffer o. 3 ~ NaC1 in T r i s - E D T A buffer i M NaCI in T r i s - E D T A buffer 7-9 inolal CsCI in T r i s - E D T A buffer
B u o y a n t d e n s i t y (g/cm 3) 25 ° , not annealed
Annealed • h at 38°
.4 n n e a l e d • h at 6 0 '
1.7o 9 1.7o 3 1.7o 7 1.7o 9
1.7o 9 1.7Ol 1.7o 5 1.7o8
1.709
1.699 1.7o2 1.703
Tile reassociation of mouse satellite DNA was examined in buffers of different ionic strengths at 25 °, 38° and 6o ° (Table V). Like other natural DNA's, and unlike poly(A) and poly(U), there was less rewinding of mouse satellite DNA at higher ionic strengths and lower temperatures. This is in accord with earlier results which indicate that separated strands of mouse satellite DNA show significant intrastrand folding under appropriate conditions 1°. No significant rewinding of natural DNA's occurs in concentrated CsCI due to extensive intrastrand folding. In conclusion, the ionic strength dependence of the reassociation of mouse satellite DNA resembles the properties of other natural DNA's rather than those of homopolymers. Denatured mouse satellite DNA nucleated in 7.9 molal CsC1 at 25 ° for 2o h banded at a density of 1.7o 9 g/cm 3, indicating that little rewinding occurred (Table VI). When the nucleated strands were dialyzed against either low-ionic strength buffers or moderate-ionic strength (0.3) buffers at 4 ° , unfolding of tile nucleated strands occurred and the DNA rewound to the extent that it banded at about 1.7oo g/cm ~ (Table VI). Nucleated bacterial DNA's dialyzed against similar low-ionic strength buffers at 4 ° also rewind, but not when dialyzed against moderate-ionic strength (o.3) buffers 11. Apparently, single strands of mouse satellite DNA are less extensively folded than single strands of bacterial DNA's at an ionic strength of o. 3. A plausible explanation for these results is that homopolymer-like regions in mouse satellite DNA do not prevent a high degree of intrastrand folding at high ionic strengths (Table II), but they do decrease intrastrand folding at moderate-ionic strengths. When the nucleated strands in IO mM Tris-HC1 p l u s o. 3 M KC1 (pH 7.9) were heated at 78° for 5 min, they rewound further and banded at 1.698 g/cm 3. Under these conditions, intrastrand folds melted out and rewinding then occurred. When the nucleated strands in 4 mM Tris-HC1 p l u s 0.5 mM E D T A (pH 7.9) were heated at 78° for 5 min both interstrand and intrastrand bonds melted and the DNA banded at the denatured density, 1.7o 9 g/cm ~. Thus,covalent cross-linking of tile DNA did not occur in these experiments. N e u r o s p o r a crassa nuclease 37-39 was used to investigate the reassociated products formed at different ionic strengths. This nuclease has a high degree of specificity for single-stranded nucleic acids, and therefore did not attack native mouse satellite DNA (Table VI). Denatured mouse satellite was either reassociated in 0.3 M NaC1 at 60 ° or nucleated, and then dialyzed against very-low-ionic strength buffers before diB i o c h i m . B i o p h y s . A c t s , 2.59 (i972) 297 3 I z
304 TABLE
D . M . KURNIT el al. VI
N . crassa NUCLEASE DIGESTION OF REASSOCIATED MOUSE SATELLITE D N A
Mouse satellite D N A ( i o / * g / m l ) was d e n a t u r e d at p H I2.5, neutralized, and reassociated under t h e c o n d i t i o n s listed above. The reassociated D N A ' s were t h e n d i a l y z e d e x h a u s t i v e l y at 4 ~' against t h e buffers indicated. After dialysis, 2-BE D N A samples were centrifuged in a n a l y t i c a l CsCl d e n s i t y gradients. Other 2-ttg s a m p l e s of D N A were i n c u b a t e d w i t h N. crassa nuclease for 3 ° inin at 38 ° before centrifugation. All nuelease digestions were in the presence of t o hiM MgC12.
Reassociation
conditions
Dialysis buffer
Density (g/cm a) after
Units as o / N . crassa nuclease
dialysis
Density (g/cm a) after digestion with N. crassa
nuclease Native I)NA
4 mY[ Tris-HC1
plus i o mM MgCI 2 (pH 7.9) 7-9 nlolal CsC1 in T r i s - E D T A buffer, 25 ° , 20 h
1.689
5
1.689
1.7Ol
5
1.693
1.699
5
1.692
1.7oo
8
i .696
4 mM Tris HCI
plus to nlM MgCI~ (pH 7.9) or
4 mM Tris-HC1 plus 0.5 nlM E D T A (pH 7-9) 0. 3 M NaCI in T r i s - E D T A buffer, 6 0 , 2l h 7.9 mo lal CsCI in Tris--EDTA buffer, 25 '~, 2o h
4 mM Tris-.HCl
plus IO InM MgCl~ (pH 7-9)
] o m M Tris HCI
plus o. 3 1~{ KCI (pH 7.9)
gesting with N. crassa nuclease. A nuclease-resistant core was obtained which formed a hypersharp band at a density of 1.693-1.692 g/cm 3 (Table VIi. The hypersharp bands we observed when the nuclease-resistant cores were banded in CsC1 density gradients apparently reflect the formation of high-molecular-weight networks 4'4°. This agrees with the finding of BRAHIC AND FRASER a~ that a nuclease-resistant core remains after rapidly renaturing mouse DNA is digested by N. crassa nuclease. The cores we observed had higher buoyant densities and molecular weights than those isolated by BRAHIC AND }?RASER89 under different incubation conditions and from a mixture of mouse main band and satellite DNA's. Unfortunately, it was not possible to investigate the double-stranded regions of mouse satellite DNA because the strands rewound during dialysis. The core of reassociated mouse satellite DNA which was resistant to N . crassa nuclease resembled that obtained after treatment with E. coli exonuclease ia. BRAHIC AND I;RASERa~ cited unpublished evidence of a primarily exonucleolytic reaction catalyzed by N. crassa nuclease with tRNA. Nucleation in the presence of polyribonucleotides
As shown previously (Table III), mouse satellite DNA, denatured in the presence of polyribonucleotides, bound ribopolymers poorly and formed only one band when centrifuged in an analytical CsC1 density gradient. Further experiments were conducted to favor polyribonueleotide binding over nucleation by adding a Io-fold excess of ribopolymer, and by adding, or by annealing, ribopolymer separately to t3iochim. I3iophys. Acta, 259 (i972) 297-312
MOUSE SATELLITE D N A
305
each D N A strand before mixing the complementary strands in CsC1 at 25 ° (Table VII). These conditions did not prevent nucleation, since one band only was always observed. With an excess of ribopolymer, some polyribonucleotide binding was evidenced by a heavier and broader, but always single, band. From these data, we conclude that at a I : I ratio of polyribonucleotide to DNA, nearly all of the polyribonucleotide bound to one strand can be displaced by the addition of the complementary strand. At higher ratios ( I O : I ) , nucleation still occurred, although some ribopolymer remained bound. In contrast, phage DNA's and a rapidly reassociating fraction from African green m o n k e y D N A show asymmetric polyribonucleotide binding by one strand in the presence of tile complementary strand s,15. TABLE V[I
NUCLEATION OF SEPARATED STRANDS IN THE PRESENCE OF POLYR1BONUCLEOTIDES Excess polyribonucleotide indicated that the molar ratio of polyribonucleotide phosphorus to DN'A phosphorus was i0 : i; otherwise, this ratio was I : i or 2 : I. The D N A concentration was i 0 / , g / n i l in annealing experiments, and 2/*g/ml in the CsC1 gradients. Except in the annealing experiments, binding reactions with the single strands were carried out at 25 ~' in concentrated CsCI.
Order o[ addztion
Conditic~zs
Buoyant density (g/cm a)
L+poly(U), then H or L + H , t h e n poly(U)
Add to CsC1
1.715
L+poly(U) + H+poly(U,G)
Add to CsC1
1.734 (broad band)
L + e x c e s s poly(U), then H
Add to CsC1
1.721 (broad band)
Add to CsCI
1.749
L + e x c e s s poly(U), then I f + e x c e s s poly(U,G)
Anneal L + p o l y ( U ) , add annealed mixture to CsC1, then add H
Annealed in o. 3 M
(a) Anneal L + e x c e s s poly(U), add to CsCI (b) Separately, anneal H + e x c e s s poly(U,G) + e x c e s s poly(G); add to (a)
Annealed in 0.3 M
NaCI, 6o ~', t h NraCl, 6o°, 5 h
1.716 1.7I 4
It is not clear whether the relative lability of polyribonucleotide-mouse satellite D N A complexes is due to better matching of the satellite H L strand complex or to the instability of such complexes as d A . rU 4t. The salient point is that strand displacement can occur if it leads to the formation of a more stable duplex. Strand displacement is essential for D N A renaturation: regions containing intrastrand folding must be melted out before hydrogen bonding occurs during the rewinding step. While the final equilibrium favors the formation of the interstrand complexes because of their greater stability, initial destabilization of the intrastrand bonds must o c c u r for t h e r e a c t i o n to proceed. T h u s , c o n d i t i o n s w h i c h i n c r e a s e t h e l a b i l i t y of h y d r o g e n b o n d s (high t e m p e r a t u r e , l o w ionic s t r e n g t h ) f a v o r i n t e r s t r a n d o v e r intrastrand bonding.
Stoichiometry o/the reaction When one mouse satellite DNA strand was reassociated with an unequal a m o u n t of t h e c o m p l e m e n t a r y s t r a n d , t h e r e a s s o c i a t e d c o m p l e x r e f l e c t e d t h e dis-
Biochim. Biophys. Acta, 259 (1972) 297-312
306
D.M. KURNIT el al.
proportion. The data in Table VIII demonstrate that increasing the proportion of one of the interacting strands gives rise to an increased proportion of that strand in the reassociated complex. Further, reaction conditions which favor unfolding of singlestranded molecules (lower ionic strengths, higher temperatures, longer annealing times) enable still more of the excess strands to enter the reassociated complex. Presumably, this is due to the unfolding of the limiting strand so that more of its sites become available for nucleation. After short annealing times, the stoichiometry in optimal reassociation conditions was the same as in concentrated CsC1 (about 2 : I). However, as indicated by its lighter buoyant density, the complex formed in optimal reassociation conditions had a different structure with greater helical content than the complex formed in CsC1. In other words, due to extensive intrastrand folding, complexes formed in CsC1 have fewer base pairs per interstrand bond. However, these complexes are stable in concentrated CsC1 at 25 ° because of the enhanced stability of base pairs under these conditions. These results are consistent with the finding that reassociated mouse satellite DNA consists of networks of molecules, in which strands are bound to two or more complementary strands at the same timO. TAI3LE
Vlll
STOICHIOMETRY OF MOUSE S A T E L L I T E D N A
REASSOCIATION
S e p a r a t e d s t r a n d s of m o u s e s a t e l l i t e D N A were r e a s s o c i a t e d at 6 o :' i n b o r a t e buffer plus 0 . 3 M N a C 1 for t h e t i m e s i n d i c a t e d , or at 2 5 ' i n T r i s - E D T A b u f f e r plus 7 . 9 m o l a l C s C I f o r 2 0 h . T h e t o t a l D N A p h o s p h o r u s c o n c e n t r a t i o n was 3 " I O - ~ M . U n d e r t h e s e conditions, t w o p e a k s w e r e a l w a y s o b s e r v e d in a n a l y t i c a l d e n s i t y gradients. T h e s e p e a k s c o r r e s p o n d to a free s i n g l e - s t r a n d e d D N A aud to a r e a s s o c i a t e d c o m p l e x of H a n d l~ strands. T h e s t o i c h i o l n e t r y was d e t e r m i n e d b y COulparing the relative areas under the peaks.
A1olar ratio o/ H strands
Reassociation conditions
Time o[ annealing (sec)
added per L strand z : i 4 : t : i 1 : 4 4.{~ : I 4.8 : 1 4.8 : I 4.8 : i 4.8 : i 4.8 : i 4.8: t 4.8 : t I : 2. 5
7.9 7.9 7-9 7.9 0. 3 0. 3 0. 3 0.3 0. 3 0. 3 0.3 0. 3 0. 3
m o l a l CsC1, m o l a l CsC1, m o l a l CsC1, n l o l a l CsC1, M ~ a C l , 60' M NaC1, 60' M NaC1, 60 M NaC1, 60 M NaCI, 6o M NaCI, 6o' M N a C 1 , 60' M NaC1, 60' 3/[ N a C 1 , 6 0 '
z5 25 25` 25 '
--o 1o 23 300 600 3600 72oo 75 6 0 0 3600
Buoyant denszty o[ complex (g/cm s) 1.712 1.714 1.7i 5 1.7o6 1.715 1.7o 3 1.7o 4 1.7o 7 1.7o8 1.7o 9 1.7io 1.711 1.695
Stoichiometry o[ complex (H strands pet" L strand) 1.8 2.2 2. 7 -~. j 2.2 2.I 2.2 2. 4 2. 7 2.8 3.0 3.4 . 0. 5
Strand reassociation and dissociation
As indicated by the dissociation constant of io -10 M for bacterial DNA • RNA hybrids 4z-44, strand displacement is an infrequent occurrrence with well-lnatched hybrid molecules under stringent conditions (66 °, Na ~ concentration less than I M). In contrast, strand displacement occurs in honlopolymer-homopolymer systenls miBiochbn. Biophys. ,4cta,
259
(1972)
297
312
MOUSE SATELLITE D N A
307
tochondrial DNA-homopolymer A and mouse satellite DNA-homopolymer systems (refs. 45, 55 and Table VII). Since mouse satellite DNA contains significant amounts of homopolymer-like regions and renatures to give mismatched hybrids ~'2, the reversibility of strand reassociation of mouse satellite DNA was investigated. The stoichiometry of the reassociation of mouse satellite DNA at 60 ° in 0.3 M NaC1 is shown in Table VII. These results show that a single strand of mouse satellite DNA cannot be saturated by annealing it with an equimolar amount of the complementary strand. Therefore, these experiments were performed with an excess of H strands in order to ensure saturation. The H strand was used in excess because free H strand bands in CsC1 at a density (1.728 g/cm a) which is more readily distinguished from the reassociated hybrid densities (i.69-i.7I g/cm a) than is the free L strand (1.699 g/cma). The experiments shown in Fig. I consisted of annealing an unlabeled mixture of separated strands of mouse satellite DNA at a ratio of 4.8 H strands per L strand. At different times during the annealing reaction, radioactive H strands were added to the mixture which was then annealed turther, so that the total annealing time was 2.5 h. The concentration of the radioactively labeled DNA was negligible compared to the concentrations of the unlabeled strands. When radioactive H strand was pre-
A
375F
t'O
sec
0.0,0
A 1 °°''
iii 30.c
0060
225
t004s
oooo I -D
0030
E
t /A/
o
/
Ioo,,
'
Ec
'"
~
1°''°~
300
I/
~0120
~
~'
It
~oo~o
150t
" -0060
0 O06
150
I
5
10
15
20
25
30
35
FRACTIONNUMBER
40
45
I
5
10
15
20
25
30
FRACTIONNUMBER
35
40
a5
Fig. 1. L a b e l e d m o u s e s a t e l l i t e D N A was o b t a i n e d b y g r o w i n g m o u s e t i s s u e c u l t u r e cells in t h e p r e s e n c e of [ M e - a H J t h y m i d i n e for 3 daY s~a- U n l a b e l e d nlouse s a t e l l i t e D N A w a s a n n e a l e d a t 6o ° in b o r a t e buffer plus o. 3 1V[NaC1 a t a m o l a r r a t i o of 4.8 H s t r a n d s pe r L s t r a n d . A f t e r t h e a n n e a l ing t i n l e i n d i c a t e d l a b e l e d H s t r a n d s were a d d e d to t h e m i x t u r e , a n d a n n e a l i n g w a s c o n t i n u e d u n t i l t h e t o t a l a n n e a l i n g t i m e w a s 2. 5 h. The r e a c t i o n w a s s t o p p e d b y a d d i n g t h e n l i x t u r e t o c o n c e n t r a t e d CsC1 a t 25 °. The r e s u l t i n g 7.9 m o l a l CsC1 s o l u t i o n w a s t h e n c e n t r i f u g e d a t 32 ooo r e v . / m i n in a Spinco f i x e d - a n g l e Ti 65 r o t o r for 66 h a t 18 °. The t u b e s were p u n c t u r e d , a n d 0.25111l f r a c t i o n s were collected. Ash 0 nm ( C ) - O ) w a s d e t e r m i n e d w i t h a Zeiss P1Vf Q I [ s p e c t r o p h o t o m eter. R a d i o a c t i v i t y p r e c i p i t a t e d b y 5 % t r i e h l o r o a c e t i c acid ( Q - Q ) w a s d e t e r m i n e d on o.2inl a l i q u o t s from each fraction.
Biochim. Biophys. Acta, 259 (1972) 297-312
308
D.M.
K U R N I T et al.
sent from t h e beginning of t h e a n n e a l i n g reaction, it h y b r i d i z e d with L s t r a n d s to tile same e x t e n t as t h e u n l a b e l e d H s t r a n d s (Fig. I a ) . L a b e l e d H s t r a n d s a d d e d l a t e r d u r i n g the annealing r e a c t i o n showed progressively less reaction with L s t r a n d s (Figs. I b - I d ) . Therefore, at some p o i n t in their reassociation process, s t r a n d s of mouse satellite D N A become s t a b l y paired. I t is possible to e s t i m a t e a second-order r a t e c o n s t a n t (k~) for the reaction bet w e e n one L s t r a n d a n d one H s t r a n d from t h e d a t a in T a b l e V I I I . A f t e r a fraction of t h e reassociation process, N, has been c o m p l e t e d , t h e effective c o n c e n t r a t i o n s of H a n d L s t r a n d s are (PT/5.8) • ( 4 . 8 - - S N ) a n d (PT/5.8) • ( I - - N ) respectively, where PT is t h e t o t a l D N A p h o s p h o r u s c o n c e n t r a t i o n a n d S is t h e n u m b e r of H s t r a n d s p r e s e n t in t h e r e a s s o c i a t e d c o m p l e x per L s t r a n d at t h e t i m e indicated. F o r a secondo r d e r reaction, d
EPr
dt 51s'('--X)
]
PT.(4.8
=*~'5.s
SN)PT
.5-- (~--x)
(i)
where t is t h e annealing t i m e before the r a d i o a c t i v e H s t r a n d s were added. E a c h value of N was corrected for the change in S which occurs with increased annealing time. Eqn. I can be i n t e g r a t e d to yield t h e following relation: l . 4 .8 - - / (
( In ] 4~ " s - S N. I
,
.
-,u(4.s))=k, l~' . t . . 5.
(2)
F r o m T a b l e I X , t h e average value of k 2 is 6 • IO ~ 1 • mole ~ • sec-L The l i t e r a t u r e value of k 2 for mouse satellite D N A reassociated in o . i 8 M N a -~ is 1. 5 • i o ~ 1 • mole 1. sec - t (ref. 46), which corresponds to a k 2 of 3.5 " IOa 1 • mole - t • sec -1 in 0. 3 M N a + (ref. 47)- The m o s t likely e x p l a n a t i o n for t h e low k 2 value of Table I X is t h a t t h e f u r t h e r b i n d i n g of excess H s t r a n d s to the reassociated c o m p l e x is e n c u m b e r e d b y H s t r a n d s a l r e a d y in t h e complex. T h e kinetic studies p r e s e n t e d here i n d i c a t e t h a t a s t r a n d of mouse satellite D N A s t a b l y b o u n d to its c o m p l e m e n t will n o t be displaced b y a c o m p e t i n g D N A TAI3LE IX DETERMINATION
OF THE
RATE
OF RFASSOCIATION
FROM STRAND
COMPETITION
DATA
A n n e a l i n g of m o u s e s a t e l l i t e D N A w a s p e r f o r m e d as d e s c r i b e d in t h e l e g e n d t o Fig. i. I n o r d e r t o c a l c u l a t e N , i t w a s a s s u m e d t h a t N = o a t t = o, a n d N - - l a t t ~ 2. 5 h . ( I - - N ) w a s d e t e r m i n e d for e a c h v a l u e of t b y d i v i d i n g t h e f r a c t i o n of c o u n t s i n t h e r e a s s o c i a t e d c o m p l e x a f t e r t h i s a n n e a l i n g t i m e b y t h e s a m e f r a c t i o n w h e n t ~ o. T h i s v a l u e of N w a s t h e n c o r r e c t e d for the change in S which occurs with increased annealing time (Table Vlll). The total DNA phosp h o r u s c o n c e n t r a t i o n in t h e s e e x p e r i m e n t s v a r i e d f r o m 3 " i o 5 t o 3 . 5 ' lO-5 M~. Time o/annealing be/ore a d d i t i o n o/ labeled H s t r a n d (see) (t)
0
3° 60 3° o 36oo
F r a c t i o n o/ L strands u n a v a i l a b l e / o r reaction with labeled H s t r a n d (N)
Second-order rate constant (k2) d e t e r m i n e d [rom Eqn. 2 (1 • mole -1 • sec -1 • lO -2 )
0 . 0 0
- -
0.36 0.54 °'91 °'98
6
B i o c h i m . B i o p h y s . A c t a , 259 (1972) 297--312
6 4 7
MOUSE SATELLITE DNA
309
strand. The kinetics of the strand competitionlreaction are second order. The predominant reaction at equilibrium is HsL ~ H s _ I L + H , where S is the number of H strands per L strand at equilibrium. The dissociation constant, KD, is defined as follows:
KI~
[Hs tL~ [HI [HsL]
(3)
Using the relations
PT [Hs_~L][HsL] I--NNand [H] =5.8 " (4"8--SN) and substituting the appropriate values of S and N determined at t = 3600 sec, the calculated value for K n is about IO 7 M. Since the reaction has not reached equilibrium at this time, this is an estimate of the m a x i m u m limit of KD. In addition, preliminary results using this strand competition method indicate that strands nucleated in concentrated CsC1 for 6 h are stably bound.
Mouse satellite DNA--how simple? Although the nucleotide organization of mouse satellite DNA is more complicated than that of homopolymers, it is simpler than that of other natural DNA's. Mouse satellite DNA exhibits some physical properties resembling those of natural DNA's and other properties resembling those of homopolymers. It resembles homopolymers in that: (I) A significant portion of the DNA is composed of the pyrimidine tract TTTTTC and related variations 5. Polyribonucleotide binding studies confirm the presence of T-rich sequences on the H strand, and, in addition, indicate the presence of (A,C)-rich (or C-rich) sequences on the H strand. (2) The base compositions of the complementary strands are unequal, as illustrated by the large buoyant density differences of the two strands in alkaline CsC12,~. (3) The buoyant density and melting temperature of the duplex molecule are not equal to those predicted from its base composition 1-3. (4) Synthetic polyribonucleotides (and possibly natural RNA's) bound to one DNA strand m a y be displaced by the addition of the complementary DNA strand. On the other hand, mouse satellite DNA has several properties which resemble those of other natural DNA's. The base composition and melting temperature show that it is not merely a polymer of the type dA. dT. The buoyant densities of the single strands (Table I) agree with the densities calculated from the base composition of each strand by an empirical equation which relates the base composition and buoyant densities of single-stranded procaryotic DNA's. The reassociation of mouse satellite DNA is inhibited at high ionic strengths and low temperatures because of intrastrand folding, and gives rise to an essentially irreversible product.
DISCUSSION Reassociation of DNA molecules in 7.9 molal CsC1 at 25 ° involves nucleation without significant rewinding. It can be estimated n from the buoyant density of the nucleated strands that the extent of rewinding is less than 5 %- Thus, reassociation in concentrated CsC1 at 25 ° is determined by the repetition frequency of short nucleotide sequences. Mouse satellite DNA, human satellite I, the three guinea pig satellites Biochim. Biophys. Acta, 2.59 (1972) 297-312
310
D . M . KURN|T
el al.
and two calf satellites all nucleate in analytical CsC1 density gradients(7.()molal CsC1, 25 °, 2o h) TM. This contrasts with h u m a n main band D N A (unpublished observations) and procaryotic D N A ' s which do not nucleate under these conditions. However, procaryotic D N A ' s will nucleate in concentrated CsC1 at 25" over a period of days 11''~5, as will lmman main band DNA at high concentrations (unpublished observations). Mouse satellite and guinea pig satellite I I)NA apparently contain a high degree of r e d u n d a n c y due to the multiplication and subsequent divergence of short (6 13 nucleotides) sequences 5. The structure proposed for these D N A ' s is consistent with their rapid nucleation rate in CsC1 at 2 5 ' and with the observed polyribonucleotide interactions. Despite its rapid nucleation in CsC1 at e5 °, guinea pig satellite I D N A in o.I8 M Na ~ at 6o ~"gives a broad, shallow Cot curve consistent with a repeated sequence of about IO5 nucleotide pairs ~,24. In contrast, a series of bacteriophages from Bacillus stearotherm@hihts and B. subtilis with genome sizes ranging from 4" ~o4 to 2 • io ~ nucleotide pairs shows ahnost no nucleation in 7.q molal Cs('l after 2o h 4~ ~0. The rate of nucleation in CsC1 is at least a qualitative measure of complexity ("the n u m b e r of base pairs in non-repeating sequences per virus or cell for the given D N A ''1~) for procaryotic D N A ' s since Hemophilus in/lue~za DNA, which is five times larger than T 4 DNA, nucleates at about 4o (.!i~of the rate of the phage DNA TM2a. Presumably, guinea pig satellite I D N A strands nucleate more frequently than bacteriophage D N A ' s in CsC1 at z5", but fewer of the nucleations remain stable under the more restrictive conditions (6(¢, o.I8 M Na~). The nucleation of short nucleotide sequences of procaryotic D N A ' s leads to binding of proper D N A complements 19. As a result, the complementary regions adiacent to the nucleation sites are brought into apposition and provide the homology required for stability under more stringent conditions. In contrast, the nucleation of animal satellite D N A ' s inw)lves the pairing of short nucleotide sequences which are not on proper c o m p l e m e n t a r y strands; regions adiacent to the nucleation site need not be complementary. Therefore, these nucleations are not necessarily stable under more stringent conditions. In other words, the " c o m p l e x i t y " measured at z5 '~ in concentrated CsC1 and t h a t measured at 6o" in o.3 M NaC1 are not the salne: the former reflects the repetition frequency of much shorter nucleotide sequences than does the latter. The complexity of procaryotic D N A ' s measured at 6o" in o.I8 M Na ~ correlates with the complexity measured at 25 '~ in concentrated Cs('l H,~. This correlation does not apply in the case of mouse satellite and guinea pig satellite l DNA's. Unless the type of sequence homology under investigation is clearly defined, the term " c o m p l e x i t y " is ambiguous when applied to these animal satellite DNA's. These D N A ' s apparently consist of reFeated nuclcotide sequences shorter than indicated from the Cot value and which are heterogeneous and/or interspersed between non-repeated sequences ",5,24. As the degree of mismatching increases, the apparent sequence length calculated from reassociation kinetics at restrictive conditions also increases. Accordingly, guinea pig satellite I DNA, which shows more mismat ching after reassociation than mouse satellite a,a.'4, has an apparent sequence length about one hundred times greater than t h a t of mouse satellite DNA under restrictive conditions 2~. Recently, SOUTHERNs~ and SWTON a~t) McCALLUM5'2 have also concluded t h a t mismatching has a significant effect on the rate of reassociation of nucleic acids. Biochim. t4iophys..4cta, 239 (1072) 297 312
MOUSE SATELLITE D N A
311
ACKNOWLEDGEMENTS
We thank Mrs. A. Tobia for her gift of labeled mouse satellite DNA and Dr. J. Hurwitz for his gift of N . c r a s s a nuclease. These studies were supported by grants from the National Science Foundation, the American Cancer Society, and the National Institutes of Health (AI 4153). D. M. K. is a Medical Scientist Trainee supported by U.S. Public Health Service grant No. 5T 5 GM 1674. J.J.M. and C.L.S. are recipients of National Institutes of Health Career Development Awards. REFERENCES I~. E. ]3OND, W. G. FLAMM, H. E. ]3URR AND S. ]3. ~3OND, J . Mol. Biol., 27 (1967) 289. G. CORNEO, E. GINELLI, C. SOAVE AND G. BERNARDI, Biochemistry, 7 (1968) 4373. (-'. L. SCHILDKRAUT AND J. J. MAIO, Biochim. Biophys. Acta, 161 (1968) 76. M. \VARING AND R. J. ]3RITTEN, Science, 154 (1966) 79 I. E. M. SOUTHERN, Nature, 227 (197 o) 794. J. MARMUR, J . Mol. Biol., 3 (1961) 208. \.V.G. FLAMM, M. MCCALLUM ANn P. M. [[:3. WALKER, Proc. Natl. Xead. Sci. U.S., 57 (I967) 1729. 8 J. J. MAIO, .]. Mol. Biol., 56 (1971 ) 5799 S. |~.IVA, 1. BARRAI, L. CAVALLI SFORZA AND A. FALASCHI, J. Mol. Biol., 45 (1969) 367. l O W . G. FLAMM, P. M. ]3. WALKER AND M. MCCALLUM, J. Mol. Biol., 4 ° (1969) 423 . 11 }¢.. H. ROWND, P h . D . Thesis, Harvard University, C a m b r i d g e , Mass., U.S.A., 1963. i2 C. L. SCHILDKRAUT AND J. J. MAIO, J. ]Viol. Biol., 46 (1969) 305 . 13 H. KUBINSKI, Z. OPARA--KUBINSKA AND W. SZYI3ALSKI, J. Mol. Biol., 20 (1966) 313 . 14 IC BURTON AND G. ]3. PETERSEN, Biochem. J., 75 (196o) 17. 15 \ v . SZYBALSKI, H. KUBINSKI AND P. SHELDRICK, Cold Spring Harbor Syrup. Quant. Biol., 3 t (1966) I23. i6 J. HAREL, N. HANANIA, H. TAPIERO AND L. HAREL, Biochem. Biophys. Res. Commun., 33 (1968) 696. 17 J. I'V[ARMUR, |{. ]{OXVND AND C. L. SCHILDKRAUT, Prog. Nucleic dcid Res. Mol. Biol., 1 (1963) 231. 18 J. G. \VETML'R AND N. DAVIDSON, J. ~lol. Biol., 31 (1968) 349' 19 C. A. THOMAS, JR., Prog. Nucleic dcid Res. IVIol. Biol., 5 (1966) 315 . 2o S. K. NIYOGI, J. Biol. Chem., 244 (1969) 1576. 21 P. M. t3. WALKER, Prog. Nucleic dcid Res. Mol. Biol., 9 (1969) 3Ol. 22 R. \Vu, J. 2~lol. Biol., 51 (197 ° ) 5Ol. 23 G. CORNEO, E. GINELLI AND E. POLLI, f . Mol. Biol., 48 (197o) 319. 24 G. CORNEO, E. GINELLI AND E. POLLI, Biochemistry, 9 (197 o) t565 . 25 J. A. SUBIRANA AND I9. DOTY, Biopolymers, 4 (1966) 17126 L. GROSSMAN, S. S. LEVlNE AND V~T. S. ALLISON, J. Mol. Biol., 3 (1961) 47. 27 J. MARMUR AND D. LANE, Proc. Natl. ,4cad. Sci. U.S., 46 (196o) 45328 P. D. R o s s AND J. M. STURTEV.A_NT, J. ,4m. Chem. Soc., 84 (1962) 45o3 . 29 F. VV. STUDIER, f . ;'VIol. Biol., 41 (1969) 199. 3 ° F. W. STUDIER, J. 3Iol. Biol., 41 (1969) 189. 31 [{. D. BLAKE AND J. ]{. FRESCO, J. IVIol. Biol., 19 (1966) 145. 32 M. LENG AND G. FELSENFELD, J. ~/[ol. Biol., 15 (1966) 45533 J. ]3RAHMS, A. M'. MICHELSON AND lX. E. VAN HOLDE, J. 2V[ol. Biol., 15 (1966) 467 . 34 J. c. TrlRIERR AND ~f. LENG, Biochim. Biophys. Acta, i82 (1969) 57535 I). ]3. MILLAR AND M. 5~ACKENZlE, Biochim. Biophys. Acta, 204 (197 o) 82. 36 L. D. INNERS AND G. FELSENFELD, J. Mol. Biol., 5 ° (197 o) 373. 37 E. Z. RABIN AND M. J. FRASER, Can..]. Bioehem., 48 (197 o) 389. 38 S. LINN AND I. R. LEHMAN, J. Biol. Chem., 240 (1965) 1287. 39 M. BRArtlC AND M. J. FRASER, Biochim. Biophys. dcta, 240 (1971) 23. 4 ° 1. ]3. DAWlD AND D. R. W'OLSTENHOLME, Biophys. J ., 8 (1968) 65. 41 5.][. CHAMBERLIN, Fed. Proc., 24 (1965) 1446. 42 R. LAVALL~ AND G. DE HAUWER, J. Mol. Biol., 37 (1968) 269. 43 J- o. ]31SHOP, Biochem. J., 116 (197 ° ) 223 . 44 D. E. KENNELL, Prog. Nucleic dcid Res. Wlol. Biol., i i (1971) 259. 45 M. J. CHAMBERLIN AND D. L. PATTERSON, J. Mol. Biol., 12 (1965) 41o. 46 R. J. ]3RITTEN AND D. E. KOHNE, Science, 161 (1968) 529. t 2 3 4 5 6 7
Biochim. Biophys. Acta, 259 (1972) 297 312
312
D . M . KURNIT et al.
47 R. J. ~[~R1TTEN AND J. SMITH, Carnegie Institution Yearbook, 68 (1969) 378. 48 S. CORDES, H. T. EPSTEIN AND J. MARMUR, Nature, 19i ( i 9 6 i ) lO97. 49 J. MARMUR AND S. CORDES, in H. J. VOGEL, V. ~BRYSEN AND J. o . LAMPEN, ln/ormational Macromolecules, A c a d e m i c Press, N e w York, 1963, p. 79. 50 G. F. SAUNDERS AND L. L. CAMPBELL, Biochemistry, 4 (1965) 2836. 51 E. M. SOUTHERN, Nature New Biol., 232 (1971) 82. 52 W. D. SUTTON AND ~ . MCCALLUM, Nature New Biol., 232 (i97 t) 83. 53 W. G. FLAMM, P. M. B. WALKER AND M. MCCALLUM, J. Mol. Biol., 42 (1969) 441. 54 A. M. TOBIA, C. L. SCHILDKRAUT AND J. J. MAIO, Bioehim. Biophys. Acta, 246 (1971) 258. 55 P- BORST AND (9. J. C. M. ]~UTTENBERG, Biochim. Biophys..4eta, 19o (i969) 391.
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