Mutation Research, 232 (1990) 141-153

141

Elsevier MUT 04891

D N A polymerase alpha from HeLa cells synthesizes D N A with high fidelity in a reconstituted replication system Michael P. Carty a, Yukio Ishimi b,,, Arthur S. Levine a and Kathleen Dixon a a Section on Viruses and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 and b Department of Molecular Biology, Sloan-Kettering Cancer Center, New York, N Y 10021 (U.S.A.)

(Received 2 October 1989) (Revision received 22 March 1990) (Accepted 23 April 1990)

Keywords: DNA polymerase alpha; DNA synthesis; DNA replication system; Replication system, reconstituted

Summary To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5'--+ 3' exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate D N A with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.

Cellular DNA replication occurs with very high fidelity; measurement of spontaneous mutation rates in mammalian cells suggests that the average

*Present address: Nucleic Acids Research, Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya Machida-shi, Tokyo 194 (Japan). Correspondence: Dr. Michael P. Carry, National Institutes of Health, Building 6, Room 1 A15, Bethesda, MD 20892 (U.S.A.).

error rate for misincorporation of bases is about 1 per 10 9 base pairs replicated (Drake, 1969). DNA polymerase alpha and DNA polymerase delta have been implicated as the major replicative DNA polymerases for mammalian chromosomal DNA (Dresler and Frattini, 1986; Hammond et al., 1987; Wong et al., 1989). The fidelity of DNA synthesis on a primed DNA template by these DNA polymerases in vitro has been measured by both forward and reversion assays (Cotterill et al., 1987;

0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

142

Fry and Loeb, 1986; Kunkel, 1985a,b; Kunkel and Alexander, 1986; Kunkel and Loeb, 1981; Kunkel et al., 1987; Loeb and Kunkel, 1982; Perrino and Loeb, 1989; Radman and Wagner, 1986; Reyland and Loeb, 1987). Although DNA polymerase delta appeared to be relatively accurate (Kunkel et al., 1987), most preparations of DNA polymerase alpha were found to be highly inaccurate (Kunkel, 1985; Kunkel and Loeb, 1981). For example, when the fidelity of polymerase alpha (Kunkel, 1985; Kunkel and Alexander, 1986) was measured in a gap filling reaction by a forward mutation assay, it was found to make about 1 error per 4000 bases incorporated (Kunkel and Alexander, 1986). This level of accuracy is far too low to account for the high level of accuracy observed during DNA replication in vivo. Although other processes such as mismatch repair likely contribute to the low error rate in vivo, this contribution is estimated by extrapolation from results in prokaryotic systems to be no more than 1 in 103 base pairs replicated (Radman and Wagner, 1986), too little to compensate for the apparent inaccuracy of the isolated DNA polymerase alpha. The development of crude in vitro DNA replication systems which will completely replicate exogenous simian virus 40 (SV40) DNA in the presence of SV40 T antigen (Li and Kelly, 1984; Stillman and Gluzman, 1985; Wobbe et al., 1985) has allowed a biochemical analysis of the process of mammalian DNA replication. It has been shown that in these systems complete viral DNA replication is dependent on both DNA polymerase alpha and a proliferating cell nuclear antigen(PCNA-)dependent activity (presumably DNA polymerase delta), as well as other cellular factors present in the crude extract (Prelich et al., 1987; Wobbe et al., 1987; Wold and Kelly, 1988). Using the SV40-based shuttle vector, pZ189 (Seidman et al., 1985), Hauser et al. (1988) measured the fidelity of DNA replication in a crude CV-1 monkey cell extract in vitro. The base misinsertion frequency was found to be about 50-fold lower in the crude extract compared to that of purified DNA polymerase alpha assayed in a gap-tilting reaction system in vitro (Kunkel, 1985b; Kunkel and Alexander, 1986). A similar result was reported (Roberts and Kunkel, 1988) when an

M13-based shuttle vector containing the SV40 origin was replicated by crude extracts from HeLa cells. These results suggested that factors in the crude extract, in addition to polymerase alpha, may be important in maintaining replication fidelity. This high accuracy of the crude system might be explained if DNA polymerase delta were the major replicative polymerase in this system; however, this does not appear to be the case (Wobbe et al., 1985, 1987). Alternatively, other components of the crude DNA replication system may serve to enhance the replication fidelity of DNA polymerase alpha. Recently, a completely reconstituted SV40 DNA replication system, consisting of purified HeLa cell DNA polymerase alpha/primase, topoisomerase II, single-stranded DNA binding protein, DNA ligase, 5'--> 3' exonuclease, and ribonuclease H, as well as SV40 large T antigen, has been described (Ishimi et al., 1988). It has been shown previously (Ishimi et al., 1988) that semiconservative, bidirectional replication occurs when DNA containing the SV40 origin is replicated in the reconstituted purified system in vitro. Thus, in this reconstituted system, DNA polymerase alpha is apparently able to carry out both leading and lagging strand synthesis. Since this system can carry out complete replication of SV40 templates, it allows the analysis of the contribution that DNA polymerase alpha can make to DNA replication fidelity. Here we describe the use of the pZ189 shuttle vector to determine the fidelity of replication by DNA polymerase alpha in this reconstituted in vitro replication system, and compare this to the fidelity of gap-filling DNA synthesis by the isolated purified DNA polymerase alpha. Materials and methods

Cells and plasmids. E. coli strain MBM7070 (Seidman et al., 1985) carries the suppressible amber mutation lacZ(Am)CA7020; in the presence of isopropyl-/3-o-thiogalactoside, an inducer of the lac operon, and 5-bromo-4-chloro-3-indolyl-fl-D-galactoside, an artificial substrate for /3-galactosidase, this strain forms blue colonies if it contains an active supF suppressor tRNA gene supplied on a plasmid and white colonies if the

143

' ~ / ' ) G III

pBR322 ori

Fig. 1. Plasmids used in fidelity assays. Panel A: pZ189 with expanded viewof the supFgene; filled area indicates structural portion of the gene, upstream region includes the promotor pre-tRNA sequences. Panel B: Gapped heteroduplex formed between pZ150 and pZ150JHR2; single stranded region extends between the two EcoRI sites on pZ150JHR2 and the molecule contains a single strand nick in the pZ150JHR2 strand at the BamHI site. Numbers 49 and 164 indicate the positions of the two point mutations used to determine mutant recoveryefficiency.

suppressor is inactive. E. coli strain G717 (GM2150) is defective in the dam methylation pathway, which normally methylates adenines in the sequence GATC. This strain was used to prepare plasmid DNA lacking methylated adenine at G A T C sites. The plasmid pZ189 (Fig. 1A; Seidman et al., 1988) carries the early region of SV40 virus and amp r, supF and the pBR327 replication function from piAN7. The plasmid pZ150 (Zagursky and Berman, 1984), generously supplied by M. Berman, consists of the entire sequence of pBR322 with the origin and intragenic region of M13 phage cloned into the AhaIII site. Plasmid pZ150JHR2 was constructed by J. Hauser by cloning the supF gene (from the plasmid piAN7) into the unique EcoRI site of pZ150. All plasmids were purified by CsC1 equilibrium sedimentation by Lofstrand Labs Ltd., Gaithersburg, MD. HeLa cells were grown as described previously (Wobbe et al., 1985).

Enzymes. DNA polymerase alpha, purified by immunoaffinity chromatography from HeLa cell extracts (Wobbe et al., 1987), was kindly provided by S.-H. Lee (Sloan-Kettering Cancer Center, NY). All restriction enzymes except BamHI and DpnI were from Bethesda Research Laboratories, Bethesda, MD. BamHI and DpnI were obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN.

Replication of pZ189 in vitro. For replication in the crude extract, pZ189 DNA (230 ng) was incubated in a reaction containing 40 mM creatine phosphate (di Tris salt, pH 7.7); 7 mM MgCI2; 0.5 mM DTT; 4 mM ATP; 200 /~M CTP, G T P and UTP; 100 ffM each of dATP, dGTP, dTTP, and [a-32p]dCTP (1-10 c p m / f m o l e , Amersham); 1 /~g creatine phosphokinase; 300-400 ~tg of HeLa extract (prepared as described [Wobbe et al., 1985]); and 0.6/tg of SV40 large T antigen (Wobbe et al., 1985). In reactions using purified HeLa cell proteins, HeLa extract was replaced by 0.5 unit polymerase alpha, 0.6 unit primase, 0.14 /~g (0.12 × 10 -3 unit) D N A ligase, 200 units DNA topoisomerase II, 0.16 /xg HeLa single-stranded DNA binding protein, 0.0057/~g (0.8 unit) 5' ~ 3' exonuclease, and 0.02 fig (1.7 units) ribonuclease H, all purified from HeLa cell-free extracts as described previously (Ishimi et al., 1988; Wobbe et al., 1985). After 3 h incubation at 37 ° C, reactions were terminated by adjusting them to 15 mM EDTA, 0.2% SDS, and 200 /~g/ml proteinase K, followed by incubation at 37 ° C for 30 min. The D N A was deproteinized by addition of 4 vol. of CsC1 (refractive index = 1.41) per volume of reaction mixture. After standing at room temperature overnight, the reaction mixture was filtered through a 0.45-~tm HA filter, and desalted and concentrated by three exchanges through a Centricon 30 concentrator (Amicon Corp., Lexington, MA). Determination of mutant frequency. Plasmid DNA, purified after replication in vitro, was treated with 0.1 unit of DpnI (Boehringer Mannheim Biochemicals, Indianapolis, IN) for 60 min at 37 °C. DpnI-resistant DNA was then treated with 1.5 units of dam methylase (New England Biolabs) for 60 min at 37°C. E. coli strain MBM7070 was transformed with the replicated, remethylated D N A by the calcium chloride procedure. Transformed bacteria were plated on Luria broth plates containing 50 ~tg/ml ampicillin, and spread with 2 mg of 5-bromo-4-chloro-3-indoyl-flD-galactopyranoside (X-gal) and 12 mg of isopropyl-fl-D-thiogalactopyranoside (IPTG), to allow us to identify the total yield of transformed colonies, and to identify mutant colonies. MBM7070 is ampicillin-sensitive, and has a suppressible amber

144

mutation in the lacZ gene; in the presence of X-gal and IPTG, transformants containing pZ189 with an active supF gene and the ampicillin resistance gene make blue colonies, and white or lightblue colonies if the supF gene is inactive. Mutations that cause a loss of supF activity have now been identified at 83 of the 140 base pairs of the supF gene target (K. Dixon, unpublished). Plasmid mutants were sequenced by the chaintermination method as described previously (Hauser et al., 1988).

Preparation of gapped DNA template. The plasmid pZ150, carrying adenine methylation at GATC sites, was prepared from E. coli strain MBM7070 and then linearized with EcoRI. Plasmid pZ150JHR2, lacking adenine methylation, was prepared in E. coli strain G717 (dam) and linearized with BamHI. The linearized D N A was extracted with phenol, ethanol precipitated and resuspended in 20 #1 TE buffer, pH 8.0. For preparation of the circular double-stranded molecule having a single-stranded gap (200 bases long) in the supF target gene (Fig. 1B), linear D N A molecules (500 ng each) were mixed together in a final volume of 1 ml 57 mM sodium phosphate buffer, pH 7.2, containing 50 mM NaC1. Samples were heated to 95 ° C for 10 min to denature the DNA strands, then transferred to 6 5 ° C for 30 min to allow reannealing to occur. The samples were then transferred to ice, and desalted and concentrated by two exchanges of buffer using a Centricon 30 microconcentrator (Amicon). Under these conditions, about 50% of the D N A was converted to gapped circular double-stranded DNA (a 1 : 1 mixture of molecules with the gap in either the transcribed or nontranscribed strand of supF), while the remainder reannealed to its complementary strand to form linear pZ150 and linear pZ150JHR2. Since linear pZ150 yields white colonies upon transfection into MBM7070, it was necessary to remove the fully methylated linear pZ150 by treatment of the preparation with DpnI. The gapped molecule is resistant to cleavage by DpnI, since it contains one methylated and one unmethylated strand. The D N A was then phenol extracted, ethanol precipitated and resuspended in 50/~1 TE buffer, pH 8.0.

DNA synthesis reactions and fidelity assays with purified HeLa polymerase alpha. DNA synthesis by purified D N A polymerase alpha/primase on the gapped molecule template was carried out in 20-/~1 reactions containing 30 mM Hepes, pH 7.5; 7 mM MgC12; 1 mM dithiothreitol; 50 /~g/ml bovine serum albumin; 100 /~M each of dATP, dGTP, dTTP, and dCTP; 10 /tCi [a-32p]dCTP; 150 ng gapped molecule DNA; and 0.44 unit of HeLa cell D N A polymerase alpha/primase. For nucleotide pool imbalance experiments, the concentration of d G T P was varied from 2 /zM to 2 raM, while keeping the concentration of the other three deoxynucleoside triphosphates at 100 /~M. After incubation at 37 o C for 60 min, the reactions were terminated by addition of EDTA to 20 mM. D N A was purified by phenol extraction and ethanol precipitation and resuspended in a small volume of TE, pH 8.0. To determine the fidelity of D N A synthesis, the bacterial strain E. coli MBM7070 was transformed with a portion of the product of the polymerization reactions. As a control for the background frequency of spontaneous mutations that might occur in E. coli MBM7070 transformed with filled gapped pZ150JHR2 plasmid DNA, a hemimethylated nicked circular pZ150JHR2 was constructed as follows: Fully methylated pZ150JHR2 was linearized using HindIII, which cuts the molecule at a single site 29 base pairs on the 3' side of the supF gene. Unmethylated pZ150JHR2, prepared from E. coli strain G717, was linearized with BamHI. The linearized DNA was purified, denatured and reannealed as described for the preparation of the gapped molecule above. The reannealed products were used to transform E. coli MBM7070, and the mutation frequency determined. The error rate for gap filling is calculated from the mutant frequency as follows: error r a t e - - m u t a n t frequency/[(detectable sites in supF) × ( f r a c t i o n of fully filled molecules)+ (estimate of detectable sites for partially filled molecules) × (fraction of partially filled molecules)]. The number of sites at which mutations can be detected in supF is 83. The estimate for sites in partially filled molecules = 83 × 0.75. The background mutant frequency (mutants observed with the nicked construct) was not subtracted in these calculations; use of these values would lead

145 to at most a 15% decrease in calculated error rate. In order to determine the percentage of base misincorporations, occurring during gap-filling in vitro, that would be expected to be expressed as mutations in the tester bacterial strain, two hemimethylated molecules having a mismatch in the supF gene were constructed, exactly as described for the preparation of the nicked construct. These molecules had the wild-type pZ150JHR2 sequence on the unmethylated strand, while the other, fully methylated, strand had a single base alteration (JHR2T164 is G : C ~ T : A at position 164; JHR2G49 is A : T ~ G : C at position 49) in the supF gene. The resulting heteroduplexes were used to determine the percent expression of the strand having the incorrect base, in E. coli MBM7070. Results

Replication of pZ189 DNA in crude HeLa cell extracts, and in the reconstituted purified system in vitro. In order to elucidate the contribution of DNA polymerase alpha to the fidelity of DNA replication in vitro, we have replicated pZ189 shuttle vector DNA in a reconstituted replication system containing HeLa cell polymerase alpha as the sole replicative polymerase (Ishimi et al., 1988) and in a crude HeLa cell replication system (Wobbe et al., 1985) that contains both DNA polymerase alpha and delta. The amount of pZ189 DNA replication occurring in vitro was determined by quantitation of the [a-32p]dCTP incorporation into acid-insoluble material (Hauser et al., 1988). In a typical reaction, 25 pmoles of pZ189 DNA was synthesized in the purified system, compared to a yield of about 75-90 pmoles with the crude system. When the reaction products were resolved on a 1% agarose gel (containing 1 /~g/ml ethidium bromide), labeled closed circular pZ189 DNA was observed in both systems upon autoradiography of the dried gel (Fig. 2A). Determination of the fidelity of pZ189 replication in vitro. The mutagenesis target (the supF gene of E. coli) in pZ189 can be assayed for mutations by measuring the ability of the plasmids to suppress a nonsense mutation in the fl-galactosidase gene of the bacterial tester strain MBM7070. To

determine the frequency of SupF mutants after replication of plasmid DNA in vitro, plasmid DNA was purified and then treated with DpnI. This enzyme cleaves the input pZ189 DNA, which carries the bacterial methylation pattern at GATC sequences; replicated molecules are either hemimethylated or fully unmethylated, and thus resistant to cleavage by DpnI. The products of the DpnI reaction were resolved on a 1% agarose gel, and DpnI-resistant labeled DNA was observed in both the crude and the purified systems (Fig. 2B), indicating that this material had undergone at least one round of replication in vitro. The observed partial conversion of the replicated molecules to either form II or linear by DpnI treatment appears to be due to a nicking activity of the enzyme on hemimethylated molecules. DpnI-resistant DNA was then treated with dam methylase to restore the normal bacterial methylation pattern at GATC sites on the newly synthesized DNA. This step is required to prevent specific repair of mismatches on the unmethylated DNA strand by the bacterial methyl-directed mismatch repair system (Hauser et al., 1988). Methylation at GATC sites was complete since the dam methylase-treated DNA became sensitive to DpnI (Fig. 2C). Treatment with DpnI followed by dam methylase allows mutations occurring during replication of pZ189 in vitro to be recovered as phenotypic changes in the transformation assay, at an efficiency of at least 30% (Hauser et al., 1988). The frequency of plasmid mutants arising during replication in vitro was determined by transformation of E. coli strain MBM7070 with the replicated, remethylated plasmid DNA. Bacteria containing wild-type pZ189 form blue colonies on the appropriate indicator agar due to suppression of the bacterial lacZ amber mutation by the SupF tRNA suppressor of the plasmid. Colonies containing SupF- plasmid mutants are white or light blue. Table 1 shows the results of three separate experiments in which pZ189 DNA was replicated in the crude and reconstituted systems. Following replication in the crude HeLa extract, a total of 17 mutants were found after screening approximately 486000 transformants (Table 1), indicating an overall mutant frequency of about 0.0035%. This value is somewhat lower than that obtained when pZ189 is replicated in a crude CV-1 monkey cell

146

A.

B.

C.

C C P

C

P

+

+

-

Dpnl

+

P Jr"

--

--

-I-

-

-I-

+ +

Dam Dpn I

Top

II--

II-Linear - -

I i _

II--

i _

m

~i~i~i!i~i~i~!i~,i

Fig. 2. Analysis of products of pZ189 replication in vitro. Panel A: pZ189 DNA was incubated either with a crude HeLa cell extract (C) or with purified HeLa proteins (P) for 3 h at 37 o C. After purification of plasmid D N A in CsC1, aliquots were electrophoresed on a 1% agarose gel containing 1 # g / m l ethidium bromide, and radioactivity was determined by autoradiography of the dried gel. The positions of closed circular DNA (I), linear DNA and nicked circular DNA (II) are indicated for each panel. Panel B: The products of pZ189 replication shown in panel A were treated with 0.1 unit of the restriction enzyme DpnI at 37 ° C for 1 h. After phenol extraction and ethanol precipitation, the DNA was analyzed by agarose gel electrophoresis and autoradiography as described for panel A. Panel C: Dpnl-treated DNA, replicated in a crude extract (C) or with purified proteins (P), was incubated at 37 ° C for 1 h, in the presence or absence of 1.5 units of dam methylase, and then treated again (or not) with 0.1 unit of DpnI. The reaction products were analysed as described for panel A.

extract in vitro (Hauser et al., 1988). When pZ189 was replicated in the reconstituted purified system containing polymerase alpha/primase, 31 mutant colonies were identified after screening 207000 transformants (Table 1). This represents an overall mutant frequency of about 0.015%, about 4 times higher than that obtained with the crude HeLa extract. In separate control experiments, we determined that when pZ189 was incubated in vitro in the

absence of SV40 T antigen, the background mutant frequency observed in both the crude and reconstituted systems was about 0.004% (mutant frequencies in four separate determinations in the crude system were 3/79000, 3/77000, 2 / 2 5 0 0 0 and 0/25000; and in one determination in the reconstituted system was 1/25 000). These values are about 10-fold higher than that observed when pZ189 is introduced directly into E. coli MBM7070 without prior incubation in vitro (J. Hauser, per-

147 TABLE 1 MUTANT FREQUENCY IN pZ189 REPLICATED IN CRUDE HeLa CELL EXTRACTS AND IN A PURIFIED REPLICATION SYSTEM IN VITRO Expt.

Replication condition

Mutant colonies/ total colonies

Mutant frequency

Error frequency per nucleotide polymerized a

1

Crude extract Purified proteins

11/166000 18/39 000

0.006% 0.046 %

1/380000 1/54 000

7.0

Crude extract Purified proteins

5/244 000 4/75 000

0.002% 0.0053%

1/1240 000 1/470 000

2.6

Crude extract Purified proteins

1/76000 9/93000

0.0013% 0.009%

1/1900000 1/260000

7.4

2 3

Crude/ pure

a Error frequency = mutant frequency/[(0.3) (number of sites with observable mutations)]. Mutations at 83 sites in the supF gene have been detected as phenotypic changes in the bacterial transformation assay.

sonal communication), suggesting that some DNA damage occurs during in vitro incubation in the absence of DNA replication. The fact that, in the crude system, the mutant frequency observed in the presence of T antigen does not differ substantially from the background frequency in the absence of T antigen means that we can calculate only an upper limit for replication errors in the crude system. Since it is not certain that the mutations observed in the absence of T antigen would occur when DNA replication is allowed by addition of T antigen, we have not subtracted this 'background' in our calculations of error rates. The observed increase in mutant frequencies in the presence of T antigen in the reconstituted system compared with the crude system cannot be accounted for by an increase in background mutations in the reconstituted system and thus appears to be due to replication errors. Clearly, there is considerable variation in the mutant frequencies observed, using both the crude and purified systems, in the 3 repetitions of the experiment. Part of this variability certainly reflects the low mutant frequencies being measured. In addition, it is likely that the absolute mutant frequency may be influenced by small variations in extract preparation or protein purification. Despite these uncertainties, we are confident that replication in both the crude and purified systems occurs with high fidelity. Moreover, the replication fidelity appears to be higher in the crude

system than in the purified system in each of the 3 separate experiments. From these data, the error rate for replication of plasmid DNA (from an SV40 origin) in HeLa extracts, and by a replication complex containing polymerase alpha as the sole DNA polymerase, can be estimated, as described in Table 1. This value, 1 per 170 000 nucleotides incorporated with the reconstituted system, compared to 1 per 700000 nucleotides incorporated with the crude extract, represents a combined frameshift and base substitution error rate for a large number of sites in the supF tRNA gene, estimated from 3 separate experiments. The fidelity of the crude HeLa extract observed here is somewhat higher than that determined previously with the same type of extract using a different vector system (1/150000; [Roberts and Kunkel, 1988]). The difference may reflect some minor variations in the method of extract preparation (Li and Kelly, 1984; Wobbe et al., 1985), or merely the variability inherent in measuring these low mutation frequencies.

Fidelity of isolated, purified DNA polymerase alpha in copying the supF target gene in vitro. The error rate observed for purified DNA polymerase alpha/primase in the reconstituted system is much lower than that reported previously for DNA polymerase alpha alone in a gap-filling reaction using a forward mutation assay with lacZalpha as the mutational target (Kunkel, 1985b; Kunkel and

148

Alexander, 1986). However, we needed to confirm that the type of immunoaffinity purified DNA polymerase alpha preparation used here in the

reconstituted system also exhibits low fidelity when it is tested in the absence of other enzymes in a gap-filling reaction where supF is the mutagenesis

B. 1 23456

",-200 nt A

Lane 1 2 3 Pol alpha + DNA M G G Top------~ RFr[~ Gapped Linear RFI

"-100 nt

Fig, 3. Analysis of products of gap-filling DNA synthesis by HeLa DNA polymerase alpha. (A) Gapped DNA was incubated at 37 °C for 60 min, either with our without HeLa polymerase alpha. After purification, the DNA was electrophoresed on a 1% agarose gel, containing 1/~g/ml ethidium bromide. Lane 1, Marker plasmid pZ150JHR2; Lane 2, gapped DNA incubated in the absence of polymerase alpha; Lane 3, gapped DNA incubated with polymerase. The positions of gapped, linear, Form I (dosed circular) and Form II (nicked circular) DNA are indicated. (B) After incubation with or without polymerase, gapped DNA was digested with EcoRI, and electrophoresed on a 6% denaturing polyacrylamide gel for 2 h at 1500 V. Lane 1, DNA without polymerase alpha; Lane 2, DNA with polymerase alpha; Lanes 3-6, sequencing ladder of supF tRNA gene, in the order A, T, G and C. (nt, nucleotides).

149

target. To do this we have constructed a hemimethylated heteroduplex circular plasmid molecule containing a 200-base long single-stranded gap which spans the supF gene (Fig. 1B). After incubation with DNA polymerase, the gapped D N A construct undergoes a shift in electrophoretic mobility (determined by electrophoresis on a 1% agarose gel in the presence of ethidium bromide) to that corresponding to the mobility of fully double-stranded nicked (Form II) DNA (Fig. 3A), indicating that the single-stranded gap was at least partially filled in most molecules during the reaction in vitro. The lengths of the filled portions of the gaps were determined by cutting the product DNA with EcoRI to remove the supF fragment from the construct. The D N A was then denatured and electrophoresed on a polyacrylamide gel. The two major radiolabeled products observed (Fig. 3B) correspond to a full-length copy (approximately 200 bases) of the gap containing the supF gene and a product 150 bases long (due to the presence of a strong pause site for the DNA polymerase). We estimate (by densitometry) from the relative amount of label in the two products that approximately 45% of the molecules are filled completely and about 55% are filled up to the pause site. When the gap-containing plasmid is filled with DNA polymerase alpha and then used to transform E. coli MBM7070, S u p F - mutants are observed at a frequency of 0.26% (Table 2). The background of mutations occurring in bacteria in this system was estimated by determining the

frequency of S u p F - mutants observed after transformation of E. coli MBM7070 with a hemimethylated homoduplex constructed from the supF-containing plasmid pZI50JHR2 and carrying single-strand nicks on opposite strands at positions 229 and 575. This construct would resemble a gapped molecule that had been completely filled. The unfilled gapped construct was also used to transform E. coli MBM7070 either directly or after incubation in the reaction mixture in the absence of D N A polymerase alpha. When the gapped construct is filled with DNA polymerase alpha, the frequency of s u p F - mutants is increased ].5-fold above the level found with unfilled molecules and almost 7-fold above the level found with the nicked construct. That the mutations observed after filling the gapped molecules are actually due to the polymerization reaction is substantiated by the effect of nucleotide pool imbalance on mutation frequency. Nucleotide pool imbalance has been shown previously to cause an increase in DNA-synthesis errors (Loeb and Kunkel, 1982). Here we show a 2-fold increase in the frequency of S u p F - mutants when the relative concentration of d G T P in the reaction is raised 10-fold. The fact that this pool bias did not have a more dramatic effect on mutant frequency [as observed in other assay systems (Loeb and Kunkel, 1982)] is probably due to the relatively high concentration of nucleotides used in this experiment and the fact that the resulting mutant frequency is the average of many sites rather than a single site. Reducing the d G T P concentration to 2 ~M leads

TABLE 2 F I D E L I T Y OF DNA SYNTHESIS IN VITRO BY P U R I F I E D HeLa POLYMERASE ALPHA DNA construct

Enzyme

[dGTP:dNTP]

SupF- mutants/ total colonies

Mutant frequency

Nicked Nicked Gapped Gapped Gapped Gapped Gapped Gapped Gapped

_ a None _ a None HeLa HeLa HeLa HeLa HeLa

_

16/3.5 9/3.0 89/5.0 58/3.5 67/2.6 61/1.7 84/1.5 120/2.6 88/3.7

0.046% 0.031% 0.18% 0.17% 0.26% 0.35% 0.55% 0.45% 2.4%

1:1 _ pol pol pol pol pol

alpha alpha alpha alpha alpha

1:l 1:1 5 :1 10 : 1 20 : 1 0.02 : 1

X 104 x 104 X 104 x 104 x 104 X 10 4

x 104 X 10 4

x 103

a D N A was not incubated in a DNA synthesis reaction. The mutant frequency of the untreated pZ150JHR2 is 0.02-0.03%.

150 TABLE 3 RECOVERY OF SupF MUTANTS FROM pZ150JHR2 H E T E R O D U P L E X MOLECULES C O N T A I N I N G A S u p F - BASE SUBSTITUTION ON THE METHYLATED STRAND A N D A SupF + U N M E T H Y L A T E D S T R A N D Methylated SupF strand

Mismatches a

Treatment

Total colonies

JHR2T164

T :C + A :G

None

1 099

705

64

JHR2T164

T :C + A :G

DpnI

1153

609

53

JHR2T164

T :C + A :G

Mbol

1 455

952

65

JHR2G49

G :T + C :A

None

3 345

2 959

88

a The first letter of each pair indicates the base present on the SupF

to an increase of almost 10-fold in mutant frequency. From the results of the gap filling experiments we can calculate an upper hmit for the fidelity of D N A synthesis by D N A polymerase alpha under our experimental conditions. If we assume 100% efficiency of recovery of base misinsertion errors, we can calculate (see Materials and Methods) an error rate of 1 error per 28 000 bases incorporated. Reconstruction experiments (Table 3) with heteroduplex molecules containing single-base mismatches in the supF gene suggest that the efficiency of recovery is probably lower (about 60%). In these experiments, hemimethylated molecules were used to test recovery of the S u p F - mutation from the methylated strand to provide an analogue for the filled molecules where the gap had been contained in the methylated strand of a hemimethylated construct. If we use the 30% figure for efficiency of recovery used in calculations for fidelity in the reconstituted system, the error rate for D N A polymerase alpha becomes 1/8400 bases incorporated. Regardless of what method is used, it is clear that the D N A polymerase alpha has a relatively low fidelity in a gap filling reaction in the absence of other proteins.

Characteristics of SupF

mutants.

To compare the types of errors made by D N A polymerase alpha in the reconstituted system with those occurring in the crude replication system, we sequenced the SupF mutants generated during replication of pZ189 in the two systems. As shown in Table 4, mutations were primarily base substitu-

White colonies

Percent SupF

methylated strand.

tions in both the crude H e L a extract and in the reconstituted replication system. The base substitution mutations were mostly (approximately 80%) G : C --, A : T transitions in both systems, as previously reported for crude CV-1 monkey cell extracts in vitro (Hauser et al., 1988). Since the replication reactions were carried out at equimolar concentrations of all four deoxynucleoside triphosphates, the high percentage of G : C ~ A : T transitions does not reflect a pool bias effect, which can alter the frequency and the specificity of mutations during in vitro replication (Roberts and Kunkel, 1988). Furthermore, the high frequency of G : C ~ A : T transitions does not merely reflect a bias in the supF detection system; of the total of 162 different mutations observed so far in this gene, 65% are at G : C base pairs and 26% are G : C ~ A : T transitions (Kraemer and Seidman, 1989); a random distribution would predict 17%. TABLE 4 CHARACTERISTICS OF SupF MUTANTS GENERATED W H E N pZ189 IS REPLICATED IN VITRO Type of mutation

Single-base substitution Tandem double-base substitution Single-base deletion Double-base deletion Large deletion Insertion

Replication system Crude

Purified

14 (82%)

19 (63.3%)

1 (5.8%) 1 (5.8%) 1 (5.8%) -

3 (10%) 1 (3.3%) 3 (10%) 4 (13.3%)

151 Discussion

The results presented here demonstrate that DNA polymerase alpha is highly accurate when carrying out the bidirectional replication of a closed circular DNA template in a reconstituted system containing six other HeLa cell proteins and SV40 large T antigen. A direct comparison of the fidelity of immunoaffinity purified HeLa polymerase alpha in replicative and in gap-filling DNA synthesis, using the same target gene, reveals that the fidelity of this enzyme increases by at least 10-fold when it is part of a complete DNA replication system in vitro. The fidelity of DNA replication by DNA polymerase alpha/primase in the reconstituted system is about 4-fold lower than DNA replication in the crude replication system, suggesting that additional cellular factors not present in the reconstituted system may contribute to the replication fidelity of the crude system. The relatively low fidelity of gap-filling observed here for immunoaftinity purified polymerase alpha using the forward mutation assay is similar to that observed by Kunkel (1985b) for DNA polymerase alpha purified by conventional means and assayed in a forward assay with lacZalpha as the mutational target. In contrast, Reyland and Loeb (1987) observed a higher fidelity for immunoaffinity purified DNA polymerase alpha with a ~X174am3 reversion assay. It is difficult to compare the absolute levels of base misinsertion in the two types of assay, because in the reversion assay only certain types of misinsertions at a small number of sites are measured, while in the forward assay misinsertions of all types at many sites are measured. It has been shown previously that the frequency of base misinsertion varies markedly from one site to another in a given mutational target (Kunkel, 1985b). Furthermore, Roberts and Kunkel (1988) have reported that the fidelity of immunoaffinity purified DNA polymerase alpha did not differ markedly from conventionally purified enzyme when a forward mutation assay was used. What is the mechanism by which the replication fidelity of DNA polymerase alpha is increased in the reconstituted system? It is possible that the accessory replication proteins present in the reconstituted system could enhance the accu-

racy of the DNA polymerase at the nucleotide selection step, altering the ability of the polymerase to discriminate between correct and incorrect nucleotides. Precedent for such an effect exists in the T4 bacteriophage system in which it was shown that the T4 gene 32 protein (a single-strand DNA-binding protein) and the gene 45 protein enhanced base selection by the T4 bacteriophage DNA polymerase (gene 43 protein) by interacting either with the DNA (gene 32 protein) or the DNA polymerase (gene 45 protein) (Topal and Sinha, 1983). Further experiments in which specific proteins present in the reconstituted system are tested for their effect on DNA replication fidelity by DNA polymerase alpha in the gap-tilting reaction would address this question. The second step at which accessory proteins might influence replication fidelity is in the elongation step, by changing the efficiency with which a correctly or incorrectly paired 3' end will be extended by incorporation of the next nucleotide. Recently, Perrino and Loeb (1989) demonstrated that the fidelity of DNA polymerase alpha was substantially influenced by the extent to which a nascent chain containing a mismatched 3' terminus could be elongated. The efficiency of elongation was influenced by the type of mismatch, the identity of the next correct nucleotide, and the concentration of the next correct nucleotide (Perrino and Loeb, 1989). It is possible that the additional proteins present in the reconstituted system might reduce the frequency with which mismatched termini are extended, perhaps by restricting the conformational flexibility of the DNA. In this regard it is interesting to note that DNA sequence analysis of SupF- mutants generated under conditions of nucleotide pool imbalance in crude extracts reveals that these mutations are not due to an increase in the misincorporation of the biased nucleotide (dGTP), but may be due instead to an increased frequency of misincorporation at sites where the next correct nucleotide to be incorporated is the biased dGTP (data not shown). In prokaryotic systems, exonucleolytic removal of mismatched nucleotides at the 3' end of the growing chain plays a major role in enhancing DNA replication fidelity. Tha most extensively studied prokaryotic DNA polymerases, E. coli

152 D N A polymerase I and bacteriophage T4 D N A polymerase, both have intrinsic 3' ~ 5' proofreading exonuclease activities which contribute substantially to their replication fidelity (Loeb and Kunkel, 1982). In contrast, most preparations of D N A polymerase alpha isolated from mammalian sources appear to lack a 3' --* 5' exonuclease activity (Loeb and Kunkel, 1982), although a highly accurate enzyme isolated from human lymphocytes that has a 3 ' ~ 5' activity was recently described (Bialek et al., 1989). The relative inaccuracy of gap-filling D N A synthesis by the purified polymerase alpha used here suggests that this preparation also lacks this activity; however, the possibility that the enzyme contains a cryptic 3' -~ 5' exonuclease activity (such as has been found in purified polymerase alpha isolated from Drosophila melanogaster [Cotterill et al., 1987]), which is activated during D N A replication in the reconstituted system, cannot be ruled out at present. Although it has been reported that preparations of polymerase alpha may contain small amounts of polymerase delta (Lee et al., 1989), this is unlikely to contribute significantly to the fidelity observed in these experiments. In order to increase fidelity 2-fold by addition of a highly accurate polymerase, it would be necessary that this polymerase account for half of the total synthesis observed. It appears unlikely that polymerase delta accounts for a substantial portion of the polymerizing activity in the reconstituted system, since PCNA, a necessary cofactor for polymerase delta, appears to be absent from the reconstituted system (Ishimi et al., 1988); replication is not stimulated by added P C N A (Ishimi et al., 1988); and sensitivity to replication inhibitors suggests that the majority of replication observed is being carried out by an alpha-like polymerase (Ishimi et al., 1988). Since replication of pZ189 occurs in the crude H e L a cell extract with higher fidelity than in the reconstituted system, it appears that other components may be necessary, in addition to those present in the reconstituted system, to achieve the low error rate observed in the crude H e L a cell extract. Clearly, the reconstituted system does not contain all of the factors present in the crude system that are thought to play a role in D N A replication. For example, D N A polymerase delta, which has been

strongly implicated in eukaryotic D N A replication (Dresler and Frattini, 1986; H a m m o n d et al., 1987) and which contains a 3 ' ~ 5' exonuclease activity that can proofread base-substitution errors (Kunkel et al., 1987), could provide additional fidelity in the crude extract. Further comparisons between the reconstituted system and the crude system, and mixing experiments in which components of the crude system are added to the reconstituted system, may reveal additional factors that influence replication fidelity. It appears that D N A polymerase alpha can carry out D N A replication with high fidelity in a reconstituted in vitro replication system, consistent with its major role in cellular D N A replication. The mechanism by which the enzyme achieves increased fidelity during the replication process has yet to be determined. However, the availability of both crude and reconstituted D N A replication systems in which individual components can be varied should allow the identification of factors that influence replication fidelity.

Acknowledgments We thank Dr. Jerard Hurwitz for many helpful discussions and continuing advice during the course of this work, and for critically reading the manuscript. We also thank Susan McGrath, a Mt. Holyoke College summer intern in our laboratory, for her assistance in part of this work. This work was supported in part by National Institutes of Health G r a n t GM-34559 to Dr. J. Hurwitz.

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