Formation of poly(dA) · poly(dT) by Escherichia coli DNA polymerase in the presence of anthracycline antibiotics

BIOCHIMICA ET BIOPHYSICA ACTA

269

BBA 97360

FORMATION OF POLY(dA) • POLY(dT) BY E S C H E R I C H I A C O L I DNA POLYMERASE IN T H E PRESENCE OF ANTHRACYCLINE ANTIBIOTICS KENNETH

OL SON, D A N I E L L U K ~ ANI) C L I F F O R D L. H A R V E Y

Chemical Research Department, Hoffmann-La Roche Ine , Nutley, N. J. OTZlO (U S A ) ( R e c e i v e d M a y 5th, 1972 )

SUMMARY

The antibiotics nogalamycm and ruticulomycm inhibited the primed or unprimed synthesis by Escher~chm coh DNA polymerase of poly [d (A-T) ] • poly ~d (T-A) ]. These antibiotics had no effect on the synthesis of the corresponding homopolymer poly(dA) • poly(dT). In the presence of nogalamycm, Eschemch,a col~ DNA polymerase synthesized the homopolymer de novo.

INTRODUCTION

Eschertchm cola DNA polymerase I catalyzes the unpnmed ("de novo") synthesis of poly[d(A-T)~- poly[d(T-A)l in the presence of dATP and d T T P 1 while poly(dC) • poly(dG) was made de novo when only dCTP and dGTP were present 2. DNA polymerase from 2llwrococcus luteus has been reported by Burd and Wells 3 to form poly(dA) • poly(dT) de novo under certain conditions of pH. In contrast, the E. col, polymerase was found to synthesize only poly~d(A-T) j.poly[d(T-A)] under a variety of conditions. McCarter et al. 4 showed that E. cola polymerase was capable of de novo synthesis of the homopolymer poly (dA). poly (dT) when high concentrations of proflawn were used in the reaction. Nogalamycm was found to inhibit strongly the transcription by E colz DNAdependent RNA polymerase of the alternatmg polymer poly [d (A-T) 1 • poly [d (T-A) 2, but not to inhibit transcription of the homopolymer poly(dA) • poly(dT) 5. Furthermore, nogalamycm stabilized a poly Ed(A-T) j • poly[d (T-A)] hehx but had no effect on the homopolymer, thus indicating that base sequence may be important in formation of the nogalamycin-polynucleotide complex 5. We report here that nogalamycin and a closely related anthracycme antibiotic, rutlculomycm, inhibit the synthesis by E. coh DNA polymerase of poly~d(A-T)] • polyEd(T-A)l but are without effect on the formation of poly(dA) • poly(dT). In the presence of nogalamycm, dATP and d T T P were found to polymerlze de novo the formation of the homopolymers MATERIALS AND METHODS

Nogalamycm was a gift from Dr P. Beale of the Uplohn Company, while rutlculomycin was donated by Dr E. Patterson of Lederle Laboratories Solutions of each * Present address

R o c h e I n s t i t u t e of M o l e c u l a r 13Lology, N u t l e y , N J , u s A

B2ochfm B*ophys Acta, 277 (1972) 269-275

270

k ()LSON ct al

were made mmledlately before use ,it a concentration of I lllg/nll m o oI M HCI The unlabeled deoxyrlbonucleotlde triplio.~phates were purchased from P L Bioclieniical~, Inc or Schwarz/Mann. Nucleoside triphosphates labeled with 14C were obtained from Schwarz/Mann All nucleoside trIphosphates used were assayed for mono- and dlphosphate contammation by paper chronlatography with ethanol-I M anniionium acetate, p H 3 8 (7° . 3 o, v/v) on \'Vhatinan No I paper for 48 h When not honl,,geneous, the tnphosphates were repurified by DEAE-cellulose chromatography with a linear gradient of o-o 5 M triethylanmlonium bicarbonate buffer (pH 7 6) a~ d~,scribed earher 6. The E. cola DNA polymerase (Fraction IV) was obtained from Worthington Biochemical. The highly purified DNA polymerase Fraction 7 wa.~ prepared by the procedure of Jovln et alS. The specific activity of this polymerase was similar to that reported by these authors. The DNA polymerase designated fraction 7(-6)wa~ purified by the Jovin et al 7 procedure except step 6 (phosphocellulose chromatography) was omitted. All DNA polymerase preparations were stored in 5 ° % glycerol at --2o°C. No loss in activity was found after I year of storage under these condmons. The DNA polymerase units were deternnned by the p o l y l d ( A - T ) l " polyld(T-A)l~ assay*. E. coh K - I 2 DNA-dependent RNA polymerase was purchased from Miles Laboratories and assayed b y the procedure of Burgess" The standard reaction for polymerization of dATP and d T T P contained 67 mM potassium phosphate buffer (pH 7-4), 6.7 mM MgC12, 3 mM mercaptoethanol and 0.6 mM of each nucleoside trlphosphate The total volume, presence or absence of primer and incubation conditions are described in the legends. When sample.~ were acid precipitated, the reaction (0. 5 ml) was stopped by addition of 0. 5 ml of chilled distilled water and 0. 5 ml of 7 °o HC10~ The precipitate was filtered through a HA Milhpore filter (25 m m dmmeter), and the residue washed IO times with 3-ml portions of cold distilled water The filter was dried and counted in a toluene-based scintilla, on fluid with a Packard Trlcarb Scintillation Counter. Efficiency of counting under these condatlons was 7° ",, for 14C.

RESULTS

P u r i t y o / e n z y m e / o r polymer preparatzon

The effect of DNA polymerase purity was studied and the results are illustrated in Fig. I. I t was found that highly purified enzyme would not permit primed synthesis of the homopolymers poly(dA) • poly(dT) (Fig. Ia) nor poly(dC) • poly(dG) (not shown). However, if a small amount (0. 4 unit) of Fraction IV polymerase was added to the purified enzyme, the A,ae0 nm declined after a short lag, illustrating that the nucleoslde triphosphates were polymerlzing (Fig. Ib). These results confirm those of other workers l°'n that highly purified polymerase requires some factor, probably an endonuclease, for efficient synthesis of these homopolymers. In order t.o obtain E. col, polymerase for efficient synthesis of these homopolymers, we purified the DNA polymerase from one pound of E. coh B cells by the J o v m et al. 7 procedure Enzyme from Fraction 5 of this procedure was used for primed synthesis of poly(dA) • poly(dT) and poly(dC) • poly(dG). However, the synthesis stopped early and digestion of tile product was very rapid This enzyme (FracB~och~m t3~opkys..4cta, 277 (1972) 269-275

E. colt D N A POLYMERASE •

-0

.

.

.

.

.

.

.

271 .

.

o

I

-0 2

o ~-o

3

c

b

-0 4

I I

I 2

I 3

I 4

I 5

HOURS

F i g I E f f e c t of e n z y m e p u r i t y on s y n t h e s i s of p o l y ( d A ) • p o l y ( d T ) The r e a c t i o n m i x t u r e (o 5 ml) as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s E a c h s a m p l e c o n t a i n e d 2 4 n m o l e s of p o l y ( d A ) • pol y(dT) as p r i m e r The s a m p l e s were i n c u b a t e d a t 37°C a n d t h e A zs. nm d e t e r m i n e d a t 3 o - m m i nt e rv a l s an c u v e t t e s w i t h a I - r a m l i g h t p a t h w i t h a Gilford s p e c t r o p h o t o m e t e r , a, h i g h l y p u r i f i e d D N A p o l y m e r a s e f r a c t i o n 7 (9 6 u n i t s ) , b, D N A p o l y m e r a s e f r a c t i o n 7 (9 6 u n i t s ) plus p o l y m e r a s e f r a c t i o n IV (o 4 u n i t s ) , c, D N A p o l y m e r a s e f r a c t i o n 7 ( - - 6 ) . 9.1 u m t s

tion 5) was separated on Sephadex G-ioo (step 7 of the above workers procedure) which removes exonuclease III. Kinetics of primed poly(dA) • poly(dT) polymerization by this enzyme, polymerase 7 (-6), showed rapid net synthesis followed by slow digestion of the product (Fig. IC).

Inhtbztzon o/poly[d(A-T)] • poly[d(T-A ) ] syntheszs w~th nogalamycin Nogalamycin mhlbited the primed synthesis of polyEd(A-T)]' polyLd(T-A)] (Table I). The addition of 20 Fg of nogalamycin per ml completely inhibited the formation of this alternating polynucleotide. Ruticulomycin, a closely related but disTABLE I INHImTION OF POLYEd(A-T)] • PoLY~d(T-A)] SYNTHESIS WITH NOGALAMYClN The r e a c t i o n m i x t u r e is d e s c r i b e d m M a t e r i a l s a n d M e t h o d s The d A T P c o n t a i n e d 38 c p m z4C p e r n m o l e All s a m p l e s h a d 7 5 n m o l e s of p o l y E d ( A - T ) ] - p o l y l d ( T - A ) l p e r ml as p r i m e r a n d a t o t a l v o l u m e of o 5 ml The r e a c t i o n m i x t u r e was i n c u b a t e d 3 ° m m a t 37°C w i t h 8 2 u m t s of D N A p o l y m e r a s e 7 (--6 ) The p r o d u c t w a s acid p r e c i p i t a t e d a n d t h e radaoactavaty d e t e r m m e d as d e s c r i b e d m Nfatertals a n d 1Vfethods

Inhibitor (pg/ml)

nmoles dA T P zncorpor ated

Inh,bzt*on (%)

~qogalamycm O

IOI

O

O I

9 8

3

o 5

75 46

26 54 96

i o 20 20 O

42 O

t~utaculomycm 5 o

1.2

20

O

O

IOO

99 IOO

Bzochzm. Bzophys Acta, 277 (I972) 269-275

272

K. OL'~ON C[ a/

tingmshable anthracychne 1''1a, also wa.~ found to prevent svnthe~ls ot polv d(A T) poly[d(T-A)l. Although nogalanlycm at a concentration of 2o/~g/ml totally mhltnted ,vnthesis of alternating polyld(A T)J • poly[d(T-A)l, this amount of antlblotw had no effect on the kinetics of the homopolymer poly(dA) • poly(dT) formation (l:lg 2) Similar kinetics were obtained in the presence of 2o/2g of ruticulomycln per ml

-01

-02

cfi - 0 5

o

/ ° b

-04

t

I I

I 2

t

I

3

4

I 5

HOURS

Fig. 2. Effect of n o g a l a m y c m on k m e t m s of poly(dA) • poly(dT) synthesis The enzyme m i x t u r e (Materials and Methods) contained 42 nmoles poly(dA) • poly(dT) pmmer, 8 2 u m t s D N A polymerase f r a c t m n 7 and o 5 u m t of f r a c t m n IV polymerase An a h q u o t was placed m a c u v e t t e w i t h a I m m h g h t p a t h The .4..,0 nrn w a s determined c o n t i n u o u s l y w~th a Gflford Model 2ooo m a i n t a i n e d at a c o n s t a n t t e m p e r a t u r e of 37°C a, control reaction, b, r e a e t m n with 2 o / t g / m l nogalamycm

De novo syntheszs o / p o l y ( d A ) • poly(dT) zn presence o / n o g a l a m y c m E. colt DNA polymerase ~ynthesized poly(dA), poly(dT) in the absence ot

primer in a reaction containing 20/~g of nogalamycm per ml (Table II). Furthermore, TABLE II P R O D U C T F O R M E D IN PRESENC]~ A N D A B S E N C E

OF N O G A L A M Y C I N

E a c h t u b e contained 5 ml of reaction m i x t u r e (Materials and Methods) The m i x t u r e was menb a t e d at 37°C w i t h 16 5 u m t s of D N A polymerase 7 (--6) per ml W h e n p r i m e r was used at was the alternating p o l y m e r p o l y [ d ( A - T ) j p o l y [ d ( T - A ) J The A 2 6 n a m was followed until m m l m a t (Samples i and 3 at 24 h, Sample 2 at i h 2o man). The reaction wa~ stopped b y addltton ot I 25mlof 2MNaClpluso i mloio5MEDTAandheatlngto65°Cfor 1 5 r a m The p r o d u c t w a ~ dlalysed 46 h against I 1 of i M NaC1 which w a s changed once The sample was daalysed a n o t h e r 46 h a g a i n s t i 1 of o 05 M NaC1 and changed once. The compomOon of the p r o d u c t was determined b y u l t r a c e n t n f u g a t l o n and t e m p l a t e activity w i t h R N A polymerase

Sample

1I Ill

Nogalamycm

(20 ,ug/ml)

Pr*mer (7.5 nmoles/ml)

--

+

q-

+

Btochzrn Btophys A~ta, 277 (1972) 269-275

Product poly(dA) poly(dT) polyFd(A-T) 7_ poly[d (Y A) poly(dA) poly(dT)

E. coli D N A POLYMERASE

273

the presence of a poly[d(A-T)] • poly[d(T-A)] primer m no way effected the outcome of the reaction. The homopolymer was formed after a long lag (24 h) in the presence of poly[d(A-T)] • poly[d(T-A)] and found to be reproductible. The extent of the polymerization represented the incorporation of 25 to 50 O//oof the nucleoside triphosphates added. Characterization of the product by analytical centrifugation in an alkaline CsoSQ density gradient is shown in Fig. 3. Samples I and 3 were synthesized in the

i $75

t ,~8

t at~ Hi

i 3178

J 15o

- -

F i g 3 D e n s l t y - g r a d m n t t r a c i n g s of de novo p r o d u c t f o r m e d w i t h a n d w i t h o u t n o g a l a m y c m E a c h cell c o n t a i n e d o 16 A,85 nm u n i t of p o l y n u c l e o t l d e a n d 3 7 % (w/w) of Cs2SO * a d j u s t e d to pFI 12 4 w i t h N a O H The Sp lnco Model E c e n t r i f u g e r u n a t 44 ooo r e v / m m for 20 h a t 25°C. C a l c u l a t i o n s for d e n s i t i e s were p e r f o r m e d as i n d i c a t e d b y V m o g r a d a n d H e a r s t 15 T h e p o l y n u c l e o t l d e s r u n were s a m p l e s from T a b l e I I (I, I I a n d I I I )

TABLE III CHARACTERIZATION

OF

POL¥[d(A-T)]

RNA

WITH

POLY'MERASF

The r e a c t i o n m i x t u r e (o 25 ml) c o n t a i n e d 60 m M T r l s - H C l buffer (pH 8,o), 4 mlV[ MnC1 v I 2 m M m e r c a p t o e t h a n o l , IO n m o l e s t e m p l a t e , i o o n m o l e s A T P in r e a c t i o n s c o n t a i n i n g one t r l p h o s p h a t e a n d i o o n m o l e s each of A T P a n d U T P in r e a c t i o n s w i t h b o t h t r l p h o s p h a t e s The A T P w a s l a b e l e d in each case (14oo c p m 1~C p e r nmole). T h e r e a c t i o n m i x t u r e w a s i n c u b a t e d w i t h I 5 u n i t s of R N A p o l y m e r a s e for 3 ° m m a t 37°C. The r e a c t i o n was s t o p p e d b y a c i d p r e c i p i t a t i o n a n d ra di oa c t i v i t y i n c o r p o r a t e d w a s d e t e r m i n e d as d e s c r i b e d m M a t e r i a l s a n d M e t h o d s

Template from DNA polymerzzatzon wzth Prz me r

Nogalamyc,n

Incorporation (nmoles) of [14C]A T P zn presence of ,4 T P only

A TP~- U T P

polyEd ( A - T ) ] • p o l y [ d ( T - A ) ] --

+

_L + p o l y [ d ( A - T ) J • p o l y [ d ( T - A ) ] , Miles poly(dA) • poly(dT), BIopolymers

-+

18 6

~

005 14 9 o 13 6

21.o 31 o 18-9 18.8 14 4

Bzochzm Bzophys. Acta, 277 (1972) 269-275

274

K OLSON t'l a[.

presence of the antibiotic, both revealed the characteristic double peak round when authentic poly(dA) • poly(dT) is analyzed in an alkaline gradient. The buoyant densltxes of the peaks correspond to the values obtained by Riley et al. 14 under amular conditions (poly(dA) p = 1.379 and poly(dT) p = x 450). The single peak (Fig 3, II) formed from the de novo product made in the absence ot nogalamycm (p - I 411) corresponds to the alternating polymer 1~ It would be expected that poly(dA) • poly(dT) would be transcribed by E col~ DNA-dependent RNA polymerase in the presence of only ATP while the correspondlng alternating polynucleotlde could not be transcribed. Table I I I .~hows that this was indeed true In the presence of both ATP and U T P either alternating or honlopolymer acted as template However, if only ATP wa~ used, polymerization took place only with the homopolymer poly(dA) • poly(dT). Both polymeric productb ~yntheslzed by DNA polymerase with nogalamycm served as templates for synthe.~xs of poly(rA), thua confirming the presence of the homopolymer poly(dA) • poly(dT) in these preparatlona

DISCUSSION

Nogalamycm and the closely related but different anthracycline antibiotic rutlculomycm were shown here to inhibit the synthesis by DNA polymerase of the alternating polymer polyEd(A-T)l • poly[d(T A)] while permitting synthesis of the corresponding homopolymer. Nogalamycin had been found to stablhze (raise the Tin) the helix of the alternating polymer, but had little or no effect on the homopolymer ~. Our results confirm the suggestion that base sequence determines the interaction of nogalamycin with the DNA helix. When the nucleoside triphosphates dATP and d T T P were incubated with E . coh polymerase in the presence of nogalamycln, the homopolymer duplex dA • dT was synthesized de novo, after a long lag, instead of the alternating polyEd(A-T)l • poly[d(T-A)l As had been shown by Burd and Wells a, the DNA polymer formed in the absence of template was determined b y the composition of tile reaction mixture 0omc concentration, p H and buffer). However, the)" found that the polymerase from E . coh made only polyEd(A-T)] - poly[d(T-A)l de novo under a variety of conditions. Our observations with nogalamycm and those of McCarter et at. 4 with proflavln prove that this enzyme can also form poly(dA) • poly(dT) in the absence of template. Using partially purified E . coh DNA polymerase, it was possible to obtain poly(dA) ' p o l y ( d T ) consistently The synthesis of p o l y ( d A ) - p o l y ( d T ) had been carried out earlier with poly(rA) • poly(rU) or poly(dA), poly(dT) as templates 14 This procedure often resulted in contamination with the alternating polymer whmh was formed de novo in long reactions. The use of the antibiotlcs nogalamycm and rutlculomycln allowed the polymerization to yield the homogeneous polymer, poly(dA. dT)

ACKNOWLEDGMENT

We thank Dr A Nussbaum for discussions and advice Bzoch~m Blophys

.4cta, 277 (1972) 269-275

E col, D N A POLYMERASE

275

REFERENCES I H. K S c h a c h m a n , J Adler, C M R a d d m g , I R, L e h m a n a n d A K o r n b e r g , J B,ol Chem, 235 (196o) 3242 2 C. M R a d d m g , J. J o s s e a n d A K o r n b e r g , ,J B,ol Chem., 237 (1962) 2869 3 J F B u r d a n d R D Wells, J 3Iol B~ol,53 (197 ° ) 435 4 J- A McCarter, N K a d o h a m a a n d C T s i a p a h s , Can J. B,ochem , 47 (1969) 391 5 B K B h u y a n a n d C G S m i t h , Proc Natl Acad Scz U S , 54 (1965) 566 6 C L H a r v e y , T F Gabriel, E M W i l t a n d C C R m h a r d s o n , J Bzol Chem., 246 (1971) 4523 7 T M J o v l n , P T. E n g l u n d a n d L L B e r t s c h , J B,ol Chem , 244 (1969) 2996 8 C C R~chardson, C L Schfldkraut, H V A p o s h l a n a n d A K o r n b e r g , J B,ol. Chem, 239 (1964) 222 9 R R B u r g e s s , J B~ol Chem, 244 (1960)616o io V H P a e t k a u , Nature, 224 (1969) 37 ° I i M R i l e y a n d A Paul, B~ochem~stry, io (1971) 3819 12 L A Mltcher, W McCrae, W \V Andres, J A L o w e r y a n d N B o h o n o s , J Pharm Set, 53 (1964) 1139 13 T F B r o d a s k y , J Gas Chromatogr, 5 (1967) 311 14 M Riley, B M a h n g a n d IV[ J C h a m b e r h n , J Mol Bzol, 20 (1966) 35915 J V l n o g r a d a n d J H e a r s t , Fortschr Chem Org Naturstoffe, 20 (1969) 372

B,och~m Bzophys _4eta, 277 (1972) 269-275