DNA Repair Reactions by Purified HeLa DNA Polymerases and ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 25, Issue of September 5, pp. 12228-12234,1988 Printed in U.S.A.

DNA Repair Reactions by Purified HeLa DNA Polymerases and Exonucleases* (Received for publication, March 3,1988)

HAkan RandahlS, George C. Elliott, andStuart Linn From the Department of Biochemistry, University of California, Berkeley, California94720

PM2 duplex DNA substrates containing small gaps were utilized to study DNA repair reactions of extensively purified HeLa DNase V (a bidirectional double strand DNA exonuclease) and DNA polymerases 8, y (mitochondrial and extramitochondrial), and a holoenzyme, and 6 as a function of ionic strength. At 50 mM NaCl, DNase V carried out extensive exonucleolytic degradation, and &polymerase exhibited strand displacement synthesis. However, at 150 mM NaCl, the DNase appeared only to remove damaged nucleotides from DNA termini while B-polymerase catalyzed only gap-filling synthesis. When present in equimolar amounts, B-polymerase and DNase V (which can be isolated as a 1:1 complex) catalyzed more degradation than synthesis at 50 mM NaCl; however, at 150 mM NaCl a coupled very limited nick translation reaction ensued. At physiological ionic strength DNA polymerase a holoenzyme was not active upon these substrates. In 15 mM KC1 it could fill small gaps and carry out limited nick translation with undamaged DNA, but it could not create a ligatable substrate from UV-irradiated DNA incised with T4UV endonuclease. Mitochondrial DNA polymerase y was more active at 150 mM NaCl than at lower ionic strengths. It readily filled small gaps but wasonly marginally capable of stranddisplacement synthesis. The extramitochondrial form of y-polymerase, conversely, was less sensitive toionic strength; it too easily filled small gaps but was not effective in catalyzing strand displacement synthesis. Finally, DNA polymerase 6 was able to fill gaps of several to 20 nucleotides in 0.05 M NaCl, but at higher NaCl concentrations there was little activity. DNA polymerases 6 did not demonstrate strand displacement synthesis. Therefore, at physiological ionic strength, it appears that either DNA polymerase B or extramitochondrial DNA polymerase y might aid in short patch DNA repair of nuclear (or transfecting) DNAs, whereas mitochondrial y-polymerase might fill small gaps inmitochondrial DNA.

A previous report from this laboratory (1) demonstrated that HeLa DNA polymerase @ could fill small gaps in DNA andthen catalyze limited strand displacement synthesis. Moreover, HeLa DNase V, which alone could extensively degrade duplex DNA exonucleolytically,could be coupled with

* This work was supported by Grant GM30415 from the National Institutes of Health and by Fellowship 1F32 GM10668 (to G . C. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. j Present address: Dept. of Tumor Biology, Karolinska Institute, Box 60400, S-10401 Stockholm, Sweden.

DNA polymerase @ to catalyze a nick translation reaction. This latterprocess required a judicious choice of levels of the two enzymes, however, in order to suppress strand displacement synthesisby the polymerase or uncontrolled degradation by the DNase. In addition, the process could be terminated only by the presence of DNA ligase. Studies of the DNA polymerase 8-DNase V coupled reaction using damaged and incised SV40 minichromosomes in place of purified DNA showed a much more regulated reaction; there was little degradation of the chromatin DNA beyond removal of the damage, and synthesis was limited to filling of the excision gap (2). Surprisingly, these latter reactions appeared not to be regulated by the more complex nature of the chromatin substrate but ratherby the more physiological ionic strength which was being utilized to keep the chromatin structureundisrupted. The studies in this paper were aimed at learning how these two enzymes might be regulated with ionic strength such that DNase V acts only as an excision exonuclease, DNA polymerase @ does not catalyze strand displacement synthesis, and the two enzymes catalyze a coupled and limited excision/ resynthesis reaction. Similar studies were also undertaken with highly purified preparations from HeLa cells of DNA polymerase a holoenzyme, mitochondrial DNA polymerase y, extramitochondrial DNA polymerase y, and DNA polymerase 6. EXPERIMENTALPROCEDURES

Materials-Unlabeled deoxyribonucleoside triphosphates were from Sigma, and [ (Y-~'P]~TI'P, [3H]dTTP, and [met/~yl-~H]thymidine were from Amersham Corp. PM2 DNA (>go% Form I, 7,000-10,000 cpm/nmol) was isolated from phage grown on a thymidine auxotroph or a wild type of strain of Alterornonus espejiana Ba131 (3, 4). The DNA was UV-irradiated at 0 "C with 254-nm light in 10 mM TrisHCl, pH 7.5,2% glycerol at a fluence of 2 J/mz/s for 30 s. T4 DNA ligase wasobtained from Boehringer Mannheim; one unit is the amount required to change 1 nmol of "P from pyrophosphate into Norit-adsorbable material in 20 min at 37 "C (5).T4 UV endonuclease (fraction IV) (6) andNeurospora crassa endonuclease (fraction IX) (7) were prepared as described. Endonuclease activity was measured using a nitrocellulose filter procedure which retains nicked but not supercoiled PM2 [3H]DNA (4). One unit of endonuclease introduces 1pmol of nicks into Form I DNA/min at 37 'C when the substrate is saturating. HeLa DNA polymerases 0 and y were prepared as described by Krauss andLinn (8) with further purification by chromatography upon hydroxylapatite and DNA cellulose.' HeLa DNA polymerase a holoenzyme was prepared as described by Vishwanatha et al. (9). HeLa DNA polymerase 6 was the hydroxylapatite fraction described by Nishida et al. (10) and was free of other DNA polymerases. Incision of DNA-Incision by T4 UV endonuclease was carried out in 25 mM Tris-HC1, pH 7.5, 200 mM NaC1, 80 ng/ml bovine serum albumin, 0.3 mM UV-irradiated PM2 DNA (calculated as mononu-

' H. Randahl and S. Linn, unpublished data. Further purification steps based on procedures described by Mosbaugh and Linn (1) are to be published in detail.

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Effect of Ionic Strength on the Activitiesof DNA Polymerase P and DNase V-When Form I DNA is treated with N . crassa endonuclease, Form I1 molecules are produced, each of which

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FIG. 1. Effect of NaCl concentration upon activities of HeLa DNA polymerase fl and DNase V. A , PM2 [3H]DNAwas treated with N . crassa endonuclease to 0.65 nicks/genome, and then5.5-nmol aliquots of the DNA were assayed with 0.12 unit of @-polymerasea t the NaCl concentrations indicated. Blanks contained polymerase but were immediately quenched by stopping buffer. Incubation of the samples was for 60 min at 37 “C, and then thereactions were terminated by addition of stopping buffer and the samples filtered as described under “Experimental Procedures.” B, PM2 [3H]DNA was UV-irradiated at 2 J/m2/s for 40 s and incised by T4 UV endonuclease to yield an average of one nick/genome, and thenaliquots of 45 nmol were treated with 0.7 unit of DNase V a t various NaCl concentrations as described under “Experimental Procedures.” Duplicate samples were taken at the times indicated and monitored for nucleotides released. H,0.05 M NaCI; *, 0.10 M NaCI; D, 0.15 M NaCl. C,PM2 [3H]DNA (270 nmol) was UV-irradiated a t a dose rate of 2 J/m2/s for 40 s and incised by T4 UV endonuclease to an average of 1.5 nicks/genome. The DNA was divided into 6 aliquots and added to three pairs of reaction mixtures with the indicated NaCl concentration. The polymerase and nuclease reaction mixtures contained [32P] dTTP or unlabeled dTTP, respectively, in addition to unlabeled cATP, dCTP, and dGTP.DNA polymerase 0 (8.4 units) and DNase V (5units) weremixed and incubated on ice for 10 min before addition to each sample. Incubation was at 37 “Cand, a t the indicated times, aliquots were taken in duplicate and the extents of reaction determined as described under “Experimental Procedures.” A,A, 0.05 M NaCI; e, 0 , O . l O M NaC1; W, Q0.15 M NaCl.

appears to contain a gap of one or two molecules. Since these gaps contain 3’-OH primertermini,they provide suitable substrates for studying gap filling and strand displacement reactions, and such molecules weretherefore utilized to study the activity of DNA polymerase p at different concentrations of NaCl (Fig. IA). At 0.05 M NaC1, the polymerase catalyzed strand displace- bose-!%phosphateformed by a @elimination reaction) at the ment at a linear rate for at least 60 min, incorporating 3.3 3’ terminus and a pyrimidine dimer nucleotide at the 5’ dTMP residues (roughly 12 total nucleotides)/gap. Increased terminus (12). salt concentrationssuppressed the stranddisplacement activity, and at 0.15 M NaC1, only a total of 1-2 nucleotides could Y”Y B4 be incorporated per gap. Hence at this physiological ionic strength, P-polymerase appears only to catalyze gap filling. ...P$ pJ pJp$ P... To study the effect of ionic strength upon DNase V, PM2 DNA was UV-irradiated and then treatedwith phase T4 UV endonuclease. The treatment resulted in a population with This substratewas then exposed to DNase V at several NaCl approximately 1 nick/molecule which contained a baseless concentrations (Fig. 1B). With 50 mM NaCl in the reaction unsaturated sugar (presumably 2,3-didehydro-2,3-dideoxyri- mixture, degradation was extensive; 33 dTMP residues were

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released in 2 h, equivalent to more than 100 total nucleotides released. A sharp decline in activity was observed for increased NaCl concentrations, however, and with 0.15 M NaCl, only 2 thymine residues were released as a limit of the reaction. Within the limits of detectability of this protocol, this level is consistent with the removal only of the pyrimidine dimer nucleotide (and possibly the baseless sugar), but no undamaged nucleotides from the DNA. Hence, at physiological ionic strength, DNase V appears only to catalyze excision of damage from DNA termini. DNase V and DNA polymerase P can be isolated as an equimolar complex (l),suggesting that these two enzymes might function jointly to effect excision and repair synthesis in uiuo. In previous studies, such a reaction could be elicited in uncontrolled reactions in vitro, but no evidence was obtained for the enzymes actingina regulated, complexed manner (1).Since ionic strength had such a profound effect upon the enzymes individually (Fig. 1, A and B ) , the action of the two enzymes together was studied upon the incised UVirradiated substrate at several NaCl concentrations (Fig. IC). At 0.05 M NaC1, when the enzymes were added at what were estimated to be equimolar amounts, the DNase V excised nucleotides at a higher rate than P-polymerase incorporated them. However, at 0.10 or 0.15 M NaCl, the two enzymes seemed to work in concert, excising and incorporating equal numbers of nucleotides in an apparently coupled manner. Furthermore, beyond simple gap filling or excision of damage as is seen for the enzymes individually, a slow nicktranslation reaction ensued in which each enzyme appeared to enable the other to go beyond the limited reaction. The nick translation reaction was further studied by the subsequent addition of DNA ligase in order to test the extent of coupling of the polymerase and nuclease reactions (Fig. 2). In this case, PM2 DNA was treated with N . crassa DNase so that all molecules weregapped, and then these molecules were incubated with the P-polymerase and DNase V. After heating to 70 "C, these were finally treated with T4 DNA ligase.Sixty percent of the PM2 DNA molecules wassealed by the ligase, and these sealed molecules contained 0.9 [32P]dTMPresidues (roughly 3.5 total nucleotides) incorporated by the polymerase. The unsealed molecules contained roughly 0.6 [32P]dTMP residues each. By these criteria, the polymerase and DNase reactions are coupled to within 1 or 2 nucleotides in their joint actionwith the nuclease, possibly leading the polymerase reaction. Action of Mitochondrial DNA Polymerase y-DNA polymerase y is reported to have maximal activity at 0.1-0.15 M KC1 (13).To determine the effect of salt with a gapped substrate, nicks were introduced into UV-irradiated PM2 [3H] DNA with the T4 UV endonuclease, and then these nicks were extended by DNase V in low salt so as to form gaps of roughly 58 nucleotides. This DNA was used as substrate for y-polymerase at various NaCl concentrations (Fig. 3A). As opposed to DNA polymerase 6, the y-polymerase was more active with increasing NaCl concentration, thoughthe differences were not so great over the range tested. To investigate whether DNA polymerase y could utilize nicks or small gaps, UV-irradiated DNA was treated with T4 UV endonuclease and then preincubated with DNase V in 0.15 M NaCl so as to excise only the pyrimidine dimer. At 0.15 M NaCl the DNA polymerase y filled the small gaps slowly and about 3.5 [32P]dTMPresidues were inserted per gap after 180 min, possibly indicating limited strand displacement synthesis (Fig. 3B,squares). Similar observations were made with DNA containing small gaps created by the N. crassa nuclease.

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FIG. 2. Analysis by alkalinesucrose gradient sedimentation of DNA polymerase B plus DNase V reaction products after ligation. PM2 [3H]DNAwas treated with N. crmsa endonuclease to produce molecules containing 1 gap, and DNA synthesis reaction mixtures were prepared containing 24 nmol of this DNA, 3.1 units of &polymerase, 1.3 units of DNase V, and [32P]dTTP.One sample was heated immediately for 3 min a t 70 "C, made 0.6 mM in ATP, and then 10 units of T4 DNA ligase were added and the mixture was incubated for 30 min a t 37 "C. This sample did not incorporate significant amounts of [32P]dTTP butwas 56% Form I. (T4 DNA ligase is able to seal one-nucleotide gaps (l).) Two other samples were incubated with the polymerase for 2 h at 37 'C and thenheated for 3 min a t 70 "C and made 0.6 mM in ATP. To one was added 10 units of T4 DNA ligase (El) and to the other was added buffer (+), and incubation was continued for 30 min at 37 "C. All samples were then applied to 5-25% alkaline sucrose gradients and treated as described under "Experimental Procedures." The specific activities of the [3H] DNA and [32P]dTTP were 9700 and 4250 cpm/pmol, respectively.

When DNase V was present with the DNA polymerase y and the substrate nicked by the UV endonuclease, there was a lag before any polymerization occurred (Fig. 3B, open triangles). This lag wasshorter at lower concentrations of NaCl in which the DNase V was more active. As opposed to the combination of DNA polymerase P and DNase V, which, when added together, appeared to stimulate one another (Fig. lC), DNase V and y-polymerase do not appear to stimulate one another (Fig. 3B, triangles). At 0.15 M NaCl there was no difference in apparentactivity of DNase V whether y-polymerase was present or not, and the polymerase activity lagged behind the nuclease. This difference was more pronounced at lower salt concentrations,since DNase V has increased activity whereas y-polymerase has decreased activity in lower salt. In a final experiment, PM2 [3H]DNA was treated with N . crassa nuclease andthen with DNA polymerase y. After inactivating the polymerase, the samples were treated with T4 DNA ligase and analyzed by alkaline sucrose sedimenta-

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FIG.3. Utilization of gaps in PM2 [%]DNA by mitochondrial DNA polymerase y. PM2 [3H]DNA was irradiated with UV light at 2 J/mZ/s for 40 a and incised by T4 UV endonuclease to an average of 0.7 nicks/genome. A , the nicked DNA was treated with DNase Vto form gaps of roughly 58 nucleotides and thenexposed to DNA polymerase y in 0.05 M NaCl (A), 0.10 M NaCl (+), and 0.15 M NaCl (El). All measurements were in duplicate and utilized 0.3 unit of polymerase/assay. B , incised UV-irradiated PM2 [3H]DNA was treated with DNase V in 0.15 M NaCl to excise only the pyrimidine dimer and then added to a polymerase reaction mixture containing 0.15 M NaCl and 0.3 unit of y-polymerase/sample (El). Incised UVirradiated PM2 [3H]DNAwas added to reaction mixtures containing 0.15 M NaC1,0.3 unit of y-polymerase, and 0.05 unit DNase V/sample containing unlabeled (A)or 32P-labeleddTTP (A) and monitored for nuclease (A)or polymerase (A) activity. Allpoints representduplicate assays. The specific activities of the [3H]DNA and [32P]dTTPwere 9700 and 636 cpm/pmol, respectively. tion. Initially, all of the PM2 DNA wasnicked by the nuclease and after polymerization and ligation, 41% of the DNA migrated as Form I (Fig. 4).The mean of incorporation of ["PI dTMP in the Form I DNA was 0.3 [32P]dTMP/molecule,as expected from the filling of gaps of one or two nucleotides. The DNA in the Form I1 region had roughly the same degree of incorporation, except at the trailing edge where 32Pmay have been incorporated into some DNA fragments. It therefore appears that the DNA polymerase y can fill small gaps though it is not apparent why the ligation efficiencywas somewhat low. Action of the Extramitochondrial DNA Polymerase y-The filling of small gaps in DNA treated with the N. crassa nuclease by this polymerase does not vary much between 0.05 and 0.15 M NaCl (Fig. 5). About 0.8 dTMP residues were incorporated per gap at the initial rate. The time course of the reaction (Fig. 5) also shows a subsequent slow phase in which several more nucleotides are incorporated over a 3-h period, presumably by strand displacement synthesis. When the gapped DNA was treated with the polymerase, then with T4 DNAligase, and finally analyzed by alkaline sucrose gradient sedimentation, 46% of the DNA sedimented as Form I (Fig. 6). The peak fraction of Form I DNA contained 0.7 [32P]dTMPresidues/molecule, as expected for filling gaps of roughly 2-3 nucleotides. The DNA in theForm I1 region had roughly the same degree of incorporation, except at thetrailing edge where 32Pagain may have been incorporated into some DNA fragments. In summary, the activity of the extra-

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Relativedistancesedimented FIG. 4. Analysis by alkalinesucrose gradient sedimentation of mitochondrial DNA polymerase y reaction products after ligation. PM2 [3H]DNA was treated with N. crassa endonuclease followed by 1 unit of DNA polymerase y per 45-nmol aliquot. The remaining protocol was as in Fig. 2. E,l with DNA ligase; +, without ligase. The specific activities of the [3H]DNA and [32P]dTTPwere 9700 and 6250 cpm/pmol, respectively.

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FIG.5. Utilization of PM2 DNA treated with N . crassa nuclease by the extramitochondrial DNA polymerase 7 . N. crassa endonuclease was utilized to nick 125 nmol of PM2 [3H]DNA to 0.8 scissions/genome. The DNA was divided in three aliquots and added to a reaction mixture containing the indicated NaCl concentrations. DNA polymerase (2 units) was added to each aliquot, and the reactions were incubated at 37 "C. At the indicated times, duplicate 0.1-ml aliquots were monitored for nucleotide incorporation. W, 0.05 M NaC1; +,0.10 M NaC1; El, 0.15 M NaCl. The specific activity of the [32P]dTTPwas 4280 cpm/pmol. mitochondrial DNA polymerase y does not differ significantly from that for the mitochondrial form of the enzyme. Action of DNA Polymerase &-DNA polymerase 6 is an alike polymerase that has an intrinsic 3' to 5' exonuclease activity. When tested on DNA containing gaps of 20 nucleotides, the enzyme was very sensitive to ionic strength; complete gap filling was approached only at low concentrations of NaCl (Fig. 7 A ) .

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of extramitochondrial DNA polymerase y reaction product after ligation. The experiment was performed as in Fig. 2, except that 4.95 units of extramitochondrial DNA polymerase y were used. El, with DNA ligase; e, without ligase. The specific activities of the [3H]DNAand [32P]dTTPwere 9700 and 4250 cpm/pmol, respectively.

PM2 [3H]DNAwas also incised with N. crassa endonuclease and used as substrate for &polymerase (Fig. 7 B ) .A t 100-150 mM NaCl, incorporation was barely detectable and then only after prolonged incubation. At 0.05 M NaC1, somewhat less than 1 dTMP was incorporated per nick. Thus the enzyme appeared to fill small gaps only in low salt concentrations and did not appear to catalyze strand displacement synthesis. DNA nicked by N. crassa endonuclease was treated with DNA polymerase 6 and T4DNA ligase,and theproducts were analyzed by alkaline sucrose gradient sedimentation. When the DNA polymerase 6 reaction was carried out in 0.15 M NaCl, very little of Form I DNA was found, presumably due to a lack of activity of the &polymerase on this substrate. With 0.05 M NaCl present in the polymerase reaction, about 37% of the DNA sedimented as Form I (Fig. 8),and thisDNA contained roughly 1 [32P]dTMP residue/molecule. Thus, DNA polymerase 6 can fill small gaps only at hypotonic concentrations of NaC1. Action of DNA Polymerase a Holoenzyme-In a previous study with purified DNA polymerase a catalytic subunit(14), it was found that gaps of up to 65 nucleotides could bereduced to about 15-nucleotides in length, following which the DNA became refractory to further synthesis. To test whether accessory factors could confer upon this polymerase the ability to totally fill gaps, a large holoenzyme complex of a-polymerase was prepared according to theprocedure of Vishwanatha et al. (9). This complex contained 3’ + 5’ and 5’ + 3’ exonuclease activities as well as other peptides as described by those authors. At physiological ionic strength, the holoenzyme has little

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Time [min] FIG. 7. Utilization of gaps in PM2 [%]DNA by DNA polymerase 8. A , PM2 [3H]DNA was treated with UV light at a dose rate of 2 J/m2/s for 40 s and incised by T4 UV endonuclease to 1.3 nicks/ genome. The nicked DNA was treated with DNase V at 50 mM to form gaps averaging 20 nucleotides. The DNA (11.5 nmol) was then used as a substrate for 1.8 units of DNA polymerase 6 in 0.6-ml reactions containing 0.05 M NaCl (A), 0.10 M NaCl (e), or 0.15 M NaCl (El). At the indicated times, duplicate 0.1-ml aliquots were assayed for nucleotides incorporated. B, PM2 [3H]DNA was incised by N. crmsa endonuclease to 0.53 gaps/genome, and nucleotide incorporationwasmeasured as above. The specific activity of the 32P[dTTP]was 1390 cpm/pmol.

activity. However, in 15 mMKC1, the enzyme could convert PM2 DNA treated with N. crassa endonuclease to a form which could be sealed by DNA ligase (Fig. 9). In thiscase, 10 total nucleotides were incorporated per sealed nick in Form I DNA, and roughly 8 nucleotides/nick were found inthe unsealed Form I 1 DNA. This extent of incorporation implies that the exonuclease activity in the holoenzyme preparation might be able to serve in a nick translation type of reaction with the polymerase. In fact, some nucleotides are removed from Form I 1 DNA by the holoenzyme preparation. This nuclease does not appear to have a DNA excision repair capability, however, since UV-irradiated DNA treated with T4 endonuclease was notasubstrate for the holoenzyme polymerase activity (and was not sealable by DNAligase after polymerase treatment). As a final caveat, in the presence of the four dNTPs, the holoenzyme preparation could seal the filled gaps initially made by the N. crassa endonuclease without the addition of exogeneous DNA ligase (Fig.9). (DNA treated with UV light, T4 UV endonuclease, and holoenzyme wasnot a substrate for this endogenous DNA ligase.) Whether this ligase is part of the holoenzyme complex,or indeed, whether the capability to fill small gaps is conferred by a peptide of the holoenzyme complex, awaits furthercharacterization of that complex, especially at very low ionic strengths. What can be concluded is that some factor or factors exists in HeLa cells which, at

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FIG. 8. Analysis by alkaline sucrose gradient sedimentation of DNA polymerase 6 reaction product after ligation. The experiment was performed as in Fig. 2, except that 10 units of DNA polymerase 6 were used. D, with DNA ligase; 4, without DNA ligase. The specific activities of [3H]DNA and [3ZP]dTTPwere9700 and 5470 cpm/pmol, respectively.

FIG. 9. Analysis by alkalinesucrose gradient sedimentation of DNA polymerase a holoenzyme reaction product after ligation. The experiment was performed as in Fig. 2 with 10 nmol of PM2 DNA, 20 units of DNA polymerase, and T4 DNA ligase (incubated for 60 min) where indicated. Only 75% of the PM2 DNA was nicked after treatment with N . crassa nuclease. D, DNA nicked with N . crassa nuclease and treated with polymerase and T4DNA ligase; 4, nicked DNA treated with T4 DNA ligase only; un-nicked DNA treated with T4 DNA ligase only. The specific activities of the [3H] DNA and [32P]dTTP were3440 and 3100 cpm/pmol, respectively. The 32P]dTTP]appeared to be contaminated with some acid-insoluble material which remained near the top of the gradient.

.,

appears to be able to fill gaps, but not efficiently and only at hypotonic NaCl concentrations. Hence, as in many other least at hypotonic salt concentrations, allow(s) a-polymerase respects, &polymerase resembles, but is not identical to, ato fill small gaps. polymerase. How might these observations relate to the functioning of DISCUSSION these enzymes in uiuo? Under isotonic conditions, DNA poOur laboratory has pursued several studies directed at me- lymerase p or extramitochondrial y-polymerase, but not a- or thodically studying DNA excision repair with well character- &polymerases might aid in gap-filling DNA repair processes ized DNA or chromatin substrates and highly purified nu- of nuclear or transfecting DNAs. Moreover, small gaps in cleases and polymerases from HeLa cells (1,2,14). This report mitochondrial DNA might be filled by the mitochondrial yemphasizes the unanticipated degree of importance for con- polymerase.* Uncontrolled strand displacement synthesis, trolling such a simple parameter as ionic strength and points which might induce recombination or duplication events, out the profound qualitative effects alteration of such a pa- would appear most likely to be due to ,&polymerase. Gap rameter might have. filling in the absence of accessory fidelity factors by 8- or The most impressive observations were with DNA polym- possibly y-polymerase might contribute to the high rate of erase p and DNase V. At isotonic 0.15 M NaC1, these enzymes mutagenesis observed with transfecting DNAs in mammalian acted in a controlled physiologically sensible manner, either cells (15). This information, coupled with fidelity studies, independently or in a concerted nick translation reaction. In might therefore help to understand the roles of the various hypotonic conditions, where these enzymes are most active DNA polymerases and their accessory factors in both normal and abnormal DNA metabolic events in mammalian cells. and are normally characterized, they act without apparent control in anonphysiological manner. Ackmwkdgrnents-We thank Dr. Craig Nishida for providing 6This report examines the activities of all of the known polymerase, Roberta Johnson for her expert help with tissue culture, HeLa DNA polymerases in gap-filling reactions. @-Polymer- and Professor Earl Baril for his patient advice for purifying aase clearly is the most proficient at filling gaps, and at polymerase holoenzyme. physiological ionic strength its strand displacement reaction REFERENCES is retarded. The y-polymerases also appear to be capable of 1. Mosbaugh, D. W., and Linn, S. (1983) J. Biol. Chern. 258, 108filling small gaps in 0.15 M NaC1, though not in lower salt 118 concentrations. DNA polymerase LY catalytic subunit canadd nucleotides to large gaps but leaves a gap of roughly 15 While long-patch nucleotide excision DNA repair appears not to nucleotides as a final product (14). However, a-polymerase occur in mitochondria, our laboratory has detected and purified holoenzyme, prepared as described by Vishwanatha et al. (9), distinct mitochondrial forms of DNA glycosylases and AP endonuis capable of forming ligatable substrates from small gaps, but cleases which might elicit short-patch base excision DNA repair (A. only at very low salt concentrations. DNA polymerase d also E. Tomkinson, T. Bonk, N. Bartfeld, and S. Linn, unpublished data).

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HeLa DNA Polymerases

2. Evans, D. H.,and Linn, S. (1984) J. Biol. Chem. 259, 10252-9. 10259 3. Espejo, R. T., and Canelo, E. S. (1968) Virology 3 4 , 738-747 4. Kuhnlein, U., Penhoet, E. E., and Linn, S. (1976) Proc. Natl. Acad. Sci. U. S. A . 73, 1169-1173 5 . Weiss, B., Jacquemin-Sablon, A., Live, T. R., Fareed, G-C., and Richardson, C. C. (1968) J. Biol. Chem. 243, 4543-4555 6. Friedberg, E. C., and King, J. J. (1971) J. Bacteriol. 106: 500507 7. Linn, S., and Lehman, I. R. (1965) J. Biol. Chem. 240, 12941304 8. Krauss, S. W., and Linn, S. (1988) J. Cell. Physiol. 126,99-106

Vishwanatha, J. K., Coughlin, S. A., Wesolowski-Owen,M., and Baril, E. F. (1986) J. Biol. Chem. 261,6619-6628 10. Nishida, c., Reinhard, p., andLinn, s. (1988) J. Bwl.Chem. 263,501-510 11. Kato, A. C., Bartok, K., Fraser, M. J., and Denhardt, D. T.(1973) Biochim.Bwphys.Acta 308,68-78 12. Kim, J., and Linn, S. (1988) Nucleic Acids Res. 16,1135-1141 13. Knopf, K.-W., Yamada, M., and Weissbach, A. (1976) Biochemistry 15,4540-4548 14. Mosbaugh, D. W., and Linn, S. (1984)J. Biol. Chem. 269,1024710251 15. Razzaque, A., Chakrabarti, S., Joffee, S., and Seidman, M. (1984) Mol. Cell. Biol. 4, 435-441