Utilization of mismatched initiator termini by avian myeloblastosis virus DNA polymerase

225

Biochimica et Biophysica Acta, 565 (1979) 225--230

© Elsevier/North-Holland Biomedical Press

BBA 99565 UTILIZATION OF MISMATCHED INITIATOR TERMINI BY AVIAN MYELOBLASTOSIS V I R U S DNA POLYMERASE

GARY B. WEISS and BOYD K. CARR Division of Hematology-Oncology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.)

(Received March 13th, 1979) (Revised manuscript received July 12th, 1979) Key words: Avian myeloblastosis virus; Reverse transcriptase; DNA polymerase; DNA sy n thesis

Summary DNA synthesis by avian myeloblastosis virus was studied using poly(C) as template and modified oligo(dG) as primer. The addition of one noncomplementary base to the 3'-end of the primer has no important effect on synthesis. The mispaired base is incorporated into the product and the apparent K m (for primer) and the V of the reaction remain unchanged. This confirms the absence of a 3' -* 5'-exodeoxynuclease activity using a template that is transcribed faithfully rather than one that can undergo a slippage reaction.

Introduction

Avian myeloblastosis virus DNA polymerase (also called reverse transcriptase) requires a template (preferably but n o t necessarily RNA), a primer (preferably DNA), deoxynucleotide triphosphates (generally all four bases) and a divalent cation (generally Mg 2+) in order to synthesize DNA (for review, see Ref. 1). Almost all studies on the mechanism of action of this enzyme have used natural m R N A s or synthetic homopolynucleotides as templates and h o m o p o l y m e r s as primers. The AMV DNA polymerase has been frequently shown to prefer deoxy-primers to ribo-primers [1] and to use larger oligonucleotides as primers more effectively than small (chain length of 4) oligonucleotides [2,3]. Other characteristics of primers have not been studied extensively. Abbreviations: SDS, s o d i u m d o d e c y l s u l f a t e ; AMV, avian m y e l o b l a s t o s i s virus; (dG)]-~, o l i g o ( d G ) l 2-I 8 ; NTP, nucleoside triphosphate.

226 Battula and Loeb [4] have shown that the AMV DNA polymerase incorporates large numbers of noncomplementary nucleotides during polymerization and have attributed this infidelity, in part, to a lack of a 3'-* 5'-exodeoxynuclease activity [ 5]. Their methodology involved studying primers which after being complexed to the homopolymer template, poly(A), would have a mispaired initiator terminus. Not only did they find that the mispaired termini could be utilized by the enzyme, but also that the efficiency of utilization of the paired and mispaired termini was about equal in terms of amount of synthesis and size of products [5]. However, in view of the ability of this enzyme to synthesize a product larger than the template when poly(A) is used as template [3,6], poly(A) may represent a theoretically poor choice as template. As oligo(C) has been shown to be transcribed faithfully rather than to undergo a slippage reaction [3], we have reexamined this problem using poly(C) as template and modified oligo(dG) which was noncomplementary to C at the 3'-end as primer. With this system, the observations of Battula and Loeb [5] have been confirmed. Materials and Methods

RNA-directed DNA polymerase. Highly purified DNA polymerase from AMV strain BAL was prepared by the method of Kacian and Spiegelman [7]. Purification was performed by Life Science, (St. Petersburg, FL) and provided through the Office of Program Resources and Logistics (Viral Oncology, National Cancer Institute). The enzyme has been concentrated by dialysis against 50% glycerol, 0.2 M potassium phosphate (pH 7.2}, 2 mM dithiothreitol, 0.2% Triton X-100. This preparation (No. G1377) contained 267 ~g protein/ml and had an activity of 8121 U/ml. Templates and primers. The synthetic homopolymeric templates and oligodeoxynucleotide primers used in all assays were purchased from P-L Biochemical Laboratories (Milwaukee, WI). The homopolymeric template, poly(C), contained a broad distribution of sizes with an average s20,w value of 10. Tritiated block copolymers were prepared with calf thymus terminal deoxynucleotidyl transferase (P-L Biochemicals) using oligo(dG)l~-~8 (subsequently abbreviated as (dG)~) as the initiator [8,9]. All four tritiated deoxynucleoside triphosphates (New England Nuclear) could be added. The reactions were run at 37°C for 1 h and contained 60 mM sodium cacodylate (pH 6.8}, 10 mM MgC12, 0.4 mM ZnSO4 (for purines) or 1 mM CoCl2 (for pyrimidines), 1 mM [3H]dNTP, 1200 units of terminal transferase/ml and the appropriate concentration of (dG)iz to give the desired product. Reactions were stopped by addition of 1 ml 0.5 M NH4HCO3 and then dialyzed at room temperature against 0.5 M NH4HCO3 (four changes, each 1 1) and for 2 h against distilled water (1 l) to remove unreacted dNTP. Transcription reaction. The reactions comparing transcription activities of the various primers consisted of the following components: 50 mM Tris-HC1 (pH 7.9}, 50 mM KC1, 10 mM MgC12, 8 mM dithiothreitol, 100 pg/ml actinomycin D; 200 gM dGTP, 0.125 A2s0 units/ml poly(C), variable amounts of primers as specified in results, and 70 units/ml enzyme. The 50 pl reaction mixtures were incubated at room temperature and the reactions stopped after

227 5 min (to obtain initial reaction rates, V0) by the addition of ice-cold 10% trichloroacetic acid/0.2% sodium pyrophosphate solution. After 1 h on ice, the trichloroacetic-acid-precipitable material was collected by filtration through nitrocellulose filters (Millipore type HA, pore size 0.45 pm). The filters were washed with 5% trichloroacetic acid/0.1% sodium pyrophosphate, dried and the radioactivity counted by liquid scintillation spectroscopy. All reactions were done in triplicate. Blanks were run at each primer concentration and dGMP-incorporation was measured using [8-14C]dGTP (New England Nuclear). Polyacrylamide gel electrophoresis. DNA samples to be used for electrophoresis were prepared as described previously [10] using [U-~4C]-dGTP (Amersham) as label and primer concentrations at the Kin. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was carried out as described previously [ 11 ]. Results

Preparation of block copolymers Polydeoxyguanylate primers with 3H-labeled deoxynucleotides at the 3'-terminus were synthesized using terminal deoxynucleotidyl transferase [8,9]. A list of the primers studied and some of their properties are shown in Table I. Each of these synthetic primers, when electrophoresed on SDSpolyacrylamide gels, migrates as a single peak (Fig. la). The size is smaller than the tRNA marker and larger than dGTP.

Incorporation of block copolymers into newly synthesized DNA In order to demonstrate that the mispaired bases at the 3'-end of the primer were incorporated into the newly synthesized DNA, the primer and the DNA synthesized by the AMV DNA polymerase using that primer and poly(C) as template were electrophoresed on separate SDS-polyacrylamide gels. As oligo(dG)Tv should base pair perfectly with poly(C), it was used as the standard to which the other primers were compared. Fig. 1 shows the migration on the 2.2% SDS-polyacrylamide gel of both the dGT~ primer (Fig. la) and the newly synthesized [I4C]DNA as well as the 3H primer (Fig. lb). As can be seen, not TABLE I P R O P E R T I E S OF 3 H - L A B E L E D BLOCK COPOLYMERS Block copolymer

dNMP dGT~"

A280

A250

A260

A260

~-max at p H 7

Fraction * unreacted

Km (A256/m1)

V ** (%)

d Gi"~ d GI'TAT d G~-~'CI d G]-~T]d G~-T~-

2.14 1.34 1.47 1.29 2.24

0.55 0.53 0.56 0.57 0.57

1.09 1.07 1.07 1.03 1.04

253 253 253 254 254

0.118 0.262 0.230 0.275 0.106

0.0068 0.0073 0.0073 0.0088 0.0076

100 88 93 92 80

* T h e f r a c t i o n o f u n r e a c t e d d G ~ - in this p r i m e r w a s c a l c u l a t e d b y a s s u m i n g a Poisson d i s t r i b u t i o n and using c o l u m n 2 as the m e a n [ 8 , 1 2 - - 1 4 ] . * * T h e V for dGT~" is c o n s i d e r e d t o b e 1 0 0 % and t h e o t h e r values are e x p r e s s e d as t h e f r a c t i o n o f this V. This is d o n e b e c a u s e the V is a f u n c t i o n o f e n z y m e and t e m p l a t e c o n c e n t r a t i o n and varies slightly f r o m e x p e r i m e n t t o e x p e r i m e n t . T h e r e f o r e , values o b t a i n e d s i m u l t a n e o u s l y are n o r m a l i z e d t o a standard.

228 all of the primer has been incorporated into the product; most appears to be unreacted as it comigrates with the starting material. However, a small portion o f the primer now migrates more slowly and comigrates with the newly synthesized DNA. In duplicate experiments, the newly synthesized DNA does not always appear as a sharp peak and instead appears as a broad smear. Similarly, with dG~s-~C~-as primer, most of the primer remains unreacted. Less poly(dG) has been synthesized and a sharp peak of low molecular weight [14C]poly(dG) is n o t present, the product being represented by material across the first 6 cm of the gel. In this region, there has been a significant increase in 3H cpm representing the primer to which dGMP has been added. With the unreacted primer, there were only 25 cpm per gel slice in the slices from above the peak while in the gel containing the product, there were 75--100 cpm in these slices in spite of an 8-fold decrease in the peak counts. The other modified primers 15000 28S

18S

4S

12000

900(

L~I¸ o 600(

3000 L9

~I1

o

_,/

2

o o

c

400

a Ld k.-

~

3oo 0 11. re 0 (J Z

20O

0.. (.9 "13

I00

0

2 4 6 8 DISTANCE FROM ORIGIN (cm)

lO

0

0.02 PRIMER

"l

i

I

0,04

0.06

0.08

O.lO

(A256 units per ml)

Fig. 1. Size of d G ] ~ - p r i m e r a n d the p r o d u c t t r a n s c r i b e d f r o m ( d G ) ~ - p o l y ( C ) b y A M V D N A p o l y m e r a s e . (a) T h e u n r e a c t e d p r i m e r ( 1 4 2 0 0 0 c p m , spee. act. o f 1 0 0 c p m / p m o l of d G M P ) was a p p l i e d t o a 2.2% S D S - p o l y a c r y l a m i d e gel a n d r u n a t 5 m A / g e l for 9 0 rain. T h e s t a n d a r d s ( 3 H Nucleic Acid M a r k e r Set, S c h w a r z ] M a n n , O r a n g e b u r g , N Y ) w e r e e l e c t r o p h o r e s e d o n a s e p a r a t e gel a t t h e t i m e of t h e s t u d y . (b) T h e p r o d u c t w a s s y n t h e s i z e d in a 0 . 7 5 m l r e a c t i o n m i x t u r e w h i c h c o n t a i n e d t h e p r i m e r , d G ~ - , in a c o n c e n t r a t i o n o f 0 . 0 0 8 A 2 5 6 u n i t s p e r m l a n d 20 0 0 0 3H c p m p e r m l a n d [ 1 4 C ] d G T P w i t h a spec. act. o f 8.2 C i / m o l . T h e a l k a l i - t r e a t e d p r o d u c t was e l e c t r o p h o r e s e d as d e s c r i b e d a b o v e . Gel slices w e r e c o u n t e d w i t h d o u b l e label s e t t i n g s ( w h i c h h a d b e e n ) m a x i m i z e d using t h e p e a k slices f-tom similar gels a n d [ 1 4 C ] o r [ 3 H ] p o l y ( d G ) as s t a n d a r d s ) a n d n e t 3 H c p m ( o ) a n d 14C c p m (o) w e r e c a l c u l a t e d f o r e a c h gel slice (using t h e s a m e s t a n d a r d s to c o r r e c t for spill o v e r i n t o t h e o t h e r c h a n n e l ) . Fig. 2. M i c h a e l i s - M e n t e n Idnetics o f p o l y ( d G ) s y n t h e s i s d i r e c t e d b y m o d i f i e d p r i m e r - p o l y ( C ) c o m p l e x . T h e r e a c t i o n c o n d i t i o n s are d e s c r i b e d in t h e m e t h o d s s e c t i o n . T h e p r i m e r s s h o w n h e r e are d G ~ - ( a ) a n d

dG15--ETy(o).

229 gave results that are identical to those for dG~-~-~. Thus, the mispaired bases all can be incorporated into the newly synthesized DNA as seen by a shift in 3H radioactivity to higher molecular weights.

Kinetics o f DNA synthesis using block copolymers as primers The amount of DNA synthesis using each block copolymer as primer and poly(C) as template was measured at varying primer concentrations (Fig. 2). Similar results were obtained with each of the other primers. From experiments of this sort, the apparent K m and V could be obtained and these are shown in Table I. Reductions of enzyme concentration up to 100-fold do not alter these values (Kin or the V relative to dGT~) for dGT~ or dGT~AT. While dGT~ is the best primer (lowest K m and highest V), the difference between dGT~ and the other primers is minimal. Discussion

AMV DNA polymerase has been shown by Loeb and his coworkers [4] to incorporate large numbers of noncomplementary nucleotides during polymerization. They have proposed that errors in DNA synthesis might be causally related to malignant alterations [15] and have used the infidelity of DNA synthesis in vitro as a screen for potential mutagens or carcinogens [16]. In view of the potential importance of this phenomenon, many studies have been performed to further elucidate the mechanism of the high error frequency of AMV DNA polymerase [4,5,15,17--22] and other RNA t u m o r viruses [23,24]. Part of this mechanism has been attributed to the lack of the 3' -* 5'-exodeoxynuclease activity [ 5]. Using poly(A)2-v~ as template and d T ~ N T as primer, they found that the mispaired primer termini were incorporated into product and that there was no significant difference in the a m o u n t of synthesis with each o f the four primers. While these results are important, poly(A) may not be the ideal choice as template in that AMV DNA polymerase can produce a product larger than the template when oligo(A) [3], poly(A) [6] or natural mRNAs containing a poly(A)-region [11,25] are used as template. However, poly(dG) is more diffult to work with than poly(dT) and oligo(dG),2-18 can form aggregates [26]. We have restudied this problem and have confirmed and extended the work of Battula and Loeb [5] using modified oligo(dG)l~.,8 as primer and poly(C) as template. Using the four primers to which 'one' base has been added, we find that all have a very similar Km and V (Table I) and can be incorporated into the newly synthesized product (Fig. 1). These results are the same as those of Battula and Loeb [5]. They found that incorporation of dTMP was 89, 94 and 98% with dT46--C~, dT~AT, and dT46--GT as compared to d T ~ as primer [5]. They also found that the size of product was the same using matched or mismatched primer-template complexes [5]. Their reactions were carried out at 37°C for 60 min and contained 100 pM template nucleotide and 5 gM initiator nucleotide. Our conditions, besides choice of template, were slightly different in that reactions were done at room temperature with lower concentrations of template (10 ~M in nucleotide) and primer (up to 1 pM in nucleotide) and a smaller primer was used. Interestingly, although we found no important differ-

230 ences between dGr~ and the other primers, we have also found that dGr~ was a slightly better primer (higher V) suggesting that while a mispaired 3'-terminus does n o t prevent synthesis, it may be slightly less effective than the properly paired terminus. One explanation for the similar kinetic constants for the primers containing one additional base could be the presence of a large fraction (about 25%) of unreacted primer (Table I). This explanation is unlikely. If only the unreacted primer was a substrate, the Km should have been four times larger for these primers. However, as many primers can bind to one template, many primers are n o t used in the reaction. The findings of no drastic changes in Km or V with these modified primers (Fig. 2) suggests that the presence of one noncomplementary base at the 3'-terminus does not prevent the use of the primer for new DNA synthesis and confirms the work of Battula and Loeb [5].

Acknowledgements This study was supported in part by an institutional grant from the American Cancer Society (ACS IN l 1 2 A ) . We thank Dr. J.W. Beard and the Office of Program Resources and Logistics, Viral Oncology, National Cancer Institute for the AMV DNA polymerase used in these studies.

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