Proc. Nati Acad. Sci. USA Vol. 78, No. 11, pp. 7024-7027, November 1981

Alternate pathways of DNA replication: DNA I- dependent replication (temperature-sensitive /extragenic suppression) OSAMI NIWA*, SHARON K. BRYAN, AND ROBB E. MOSES Department ofCell Biology, Baylor College of Medicine, Houston, Texas 77030 Communicated by D. Nathans, July 10, 1981

ABSTRACT We have previously shown that someEscherichia proceed in the presence of a functional DNA polymerase I ac- coli [derivatives of strain HS432 (polAl, polB100, polC1026)] can tivity, despite a ts DNA polymerase III (6). replicate DNA at a restrictive temperature in the presence of a We report here that DNA replication in the parent strain polCts mutation and that such revertants contain apparent DNA becomes temperature-resistant with introduction ofDNA poly- polymerase I activity. We demonstrate here that this strain ofE. merase I activity but is ts in the absence of DNA polymerase colibecomes temperature-resistant upon the introduction ofa nor- I or presence of a ts DNA polymerase I activity. We conclude mal for DNA polymerase I or suppression of the polAl non- that this strain contains a sense mutation. Such temperature-resistant phenocopies become mutation (pcbA-) that allows repli- temperature-sensitive upon introduction of a temperature-sensi- cation to be dependent on DNA polymerase I polymerizing tive DNA polymerase I gene. Our results confirm that DNA rep- activity. This locus can be transduced to other E. coli strains and lication is DNA polymerase I-dependent in the temperature-re- again exerts phenotypic suppression of the polCts mutation in sistant revertants, indicating that an alternative pathway of the presence of DNA polymerase I. Our results indicate that replication exists in E. coli. HS432 contains a transducible locus E. coli has alternative pathways of DNA replication. (which we term pebA) that can support an alternate pathway in otherE. coli strains, so the effect ofsuppression ofpolCts is a gen- MATERIALS AND METHODS eral one. Bacterial Strains and Phage. E. coli strain HS432 (polA-, polB-, polC1026, his-, leu-) and its temperature-resistant de- On the basis ofavailable mutants and in vitro DNA replication rivative CSM61 (poLA-, polB-, polCts), and strains RK4604 systems, DNA replication in appears to be a (his-, lacam, nmtB-, metD-), E511 (polCts), and MJ245 (ValR, multistep process requiring a number ofproteins (1, 2). Ofthe metE-) have been used previously (5). FTP439 (metE+, Vals, three known DNA in that organism, DNA poly- MeMeSR) was from E. Murgola (M. D. Anderson Hospital and merase III appears to be uniquely required for replication be- Tumor Institute). Strains HS405 (polA12) and 108 (polA12) cause polCts (dnaEts) mutants, conditionally defective in rep- were from H. Shizuya (University of Southern California). lication, contain a temperature-sensitive (ts) DNA polymerase Strain E486 (polA', polC486) was from C. McHenry (University III (3). DNA polymerase I seems to be required for cell growth ofTexas Health Science Center). Phage A i21nin5psu+2(s), q480 because mutants defective in the 5' -- 3' show wild-type, and 480 psu+3 sus2 were from H. Ozeki (Kyoto much decreased viability (4). However, nonsense mutants de- University). Phage ON1 is ahybrid phage constructed by across fective in the polymerase function ofthis but containing between the two suppressor-carrying phages above, selected nearly normal levels ofthe 5' -- 3' exonuclease activity (polAl) for h+w, i2l; it carries psu+3 and attes. grow normally although replicative intermediates show slow Materials. Growth media were purchased from Difco. 3H- transition to full-size DNA in such mutants (4). Thus, DNA Labeled nucleoside triphosphates were purchased from Amer- polymerase III appears to be strictly required for synthesis of sham/Searle. N-Ethylmaleimide (MalNEt) and methyl meth- newly replicated DNA strands whereas DNA polymerase I is anesulfonate (MeMeS) were purchased from Eastman. required for a less-specific exonucleolytic function, perhaps re- Culture Methods. Cells were grown in L broth. Selection moval ofRNA primers from Okazaki pieces, and probably plays for auxotrophic markers was done on M-9 medium. MeMeS a facilitative role in elongation and ligation of nascent strands. resistance was measured as described (5). Temperature-resist- The presence ofDNA polymerase I does not change the ts char- ant phenotype was measured by growth of duplicate L-broth acter of polCts mutants. No role has been defined for DNA plates at 320C and 420C. Val resistance was measured by growth II in on M-9 plates containing L-valine at 50 jig/ml. P1 transduction polymerase replication. was done as described by Lennox (7), with mutations in the ilv We have reported (5) that one strain of E. coli containing and metE in the region ofpolA (8). Introduction ofTnlO polAl and polCts mutations shows Pol I' character in a high near pcbA was done according to Kleckner et aL (9). Phage Y- percentage of spontaneous temperature-resistant revertants. 533-cI857b221 0am29 TnlO was obtained from H. I. Miller The DNA polymerase I activity can arise by either intragenic (National Cancer Institute) and was used to infect (multiplicity reversion to polA or suppression of the polAi nonsense mu- of infection, 10) CSM61 (polAl, polB100, polC1026, pcbA-), tation by extragenic mutation. Such temperature-resistant re- a spontaneous temperature-resistant derivative of HS432 pre- vertants were demonstrated to still contain polCts by biochem- viously described (5). After 1 hr at room temperature, the cells ical and genetic tests. One interpretation of the results is that were plated on L agar containing tetracycline (15 ,ug/ml) and the strain contains a mutation that allows DNA replication to Abbreviations: ts, temperature-sensitive; MalNEt, N-ethylmaleimide; The publication costs ofthis article were defrayed in part by page charge MeMeS, methyl methanesulfonate; TetR, tetracycline-resistant. payment. This article must therefore be hereby marked "advertise- * Present address: Mitsubishi-Kasei Institute ofLife Sciences, 11, Min- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. amiooya, Machida-shi, Tokyo, Japan. 7024 Downloaded by guest on September 27, 2021 Genetics: Niwa et al Proc. Natl. Acad. Sci. USA 78 (1981) 7025

10 mM and incubated at 41°C. Cells surviving Table 2. Construction of lysogens containing suppressors had the gene for tetracycline resistance (TetR) inserted ran- MalNEtR domly about the E. coli . Five hundred independent DNA colonies were collected into 0.7% L agar as a pool of random polymerase insertions. Phage P1 was grown on this pool and used to infect activity E511 (polA+, polCts, ts). Selection for transductants- was by Lysogen phenotype (DNA growth at 42°C on L agar containing tetracycline at 25 ,ug/ml. Temp. - Individual colonies were picked and purified by repeated Recipient strain Phage resistant MeMeSR ae I), % growth under these conditions. Strain 61P-14 is one such iso- HS432 (polAl, late. Linkage of TetR and pcbA (the gene conferring tempera- polCts) 480 (su+3) 4/4 4/4 52 ture-resistant phenotype in polA+, polCts strains) was verified HS432 A (su+2) 4/4 4/4 12 by subsequent transduction (see Table 5). CSM61 (poll, DNA Polymerase Extraction and . The isolation of polCts, temp. DNA polymerase I was by Brij lysis ofEDTA/lysozyme-treated resistant) 080 (su+3) 3/3 3/3 75 cells as described (5). CSM61 None 10 RN1 (polCts, ts) ON1 (su+3) 0/5 0/5 iCO RESULTS RN1 None 100 HS432 Becomes Temperature-Resistant with polA+. To test Phage carrying suppressors were grown on strains as indicated and whether the DNA polymerase I activity arising in spontaneous stable lysogens were picked from plaque centers at 320C. Individual lysogens were tested as described. Strain RN1 is a derivative of temperature-resistant revertants of HS432 had peculiar prop- RK4604 carrying poIC1026 transduced from HS432 by using metD- erties and to assess whether the high incidence of Pol I' phe- as a marker, as described (5). The value of DNA polymerase I notype was incidental to the temperature-resistant phenotype, (MalNEtR) activity in extracts of RN1 was set as 100% (60 pmol of we transduced a wild-type polA+ gene into the strain (Table 1). dTMP incorporated). This was a direct test of the hypothesis that HS432 contains a "background" mutation permitting DNA replicative synthesis vertants arising due to suppression we observed low levels of to be dependent on DNA polymerase I and not dependent on DNA polymerase I activity, suggesting an atypical distribution DNA polymerase III at 42°C. The results demonstrate that in- ofsuppressor mutations (5). Toinvestigate whether typical non- troduction ofpolA+ converted HS432 to temperature-resistant sense suppressors give the temperature-resistant phenotype we (class III). This leads to the conclusion that HS432 can replicate used lysogenic phage carrying suppressors su+2 or su+3. With DNA at the restrictive temperature if a wild-type DNA poly- these we constructed lysogens (Table 2) picked at low temper- merase I activity is present. We verified that wild-type DNA ature. Lysogens selected were temperature-resistant and polymerase I activity was expressed by preparing extracts of MeMeSR with either suppressor. The action ofthe suppressors several of the MeMeSR transductants and measuring the level was verified by measuring DNA polymerase I levels in extracts of MalNEtR DNA polymerase activity. (approximately 50% ofnormal in su+3 lysogens and 15% in su+2 This result agrees with another observation. When we se- lysogens). Our conclusion is that "typical" suppression products lected spontaneous MeMeSR revertants (conversion to Pol I') ofpolAl allow replication at restrictive temperature. Although of HS432 we observed that 90-95% were temperature-resis- there was no reason to think that a suppressor mutation would tant. This had suggested that selection for Pol I' phenotype was have an effect on the polCts mutation, we checked by intro- associated with conversion of ts to temperature-resistant phe- ducing su+3 into a ts strain containing polCts and polA+ (RN1 notype in HS432. Our results are compatible with a single-step in Table 2). Offive lysogens picked, none was temperature-re- mutation to Pol I' (either by intragenic mutation or suppres- sistant. Thus, there is no evidence that there was an effect on sion) accountingfor the temperature-resistant phenotype, at the polC in HS432 that permitted growth at restrictive tempera- frequency at which temperature-resistant revertants arose tures. When independent lysogenized colonies were cured, (about 10-5). monitored by a lacam mutation in the strain, the strain became Suppression ofpoLAl in HS432 Produces.Temperature-Re- ts again (Table 3). sistant Phenotype. In results we reported previously (5, 6) it These results are compatible with the existence of a genetic was clear that some ofthe temperature-resistant revertants iso- variation in HS432 that permits DNA replication to be depen- lated in HS432 had a Pol I' phenotype on the basis of suppres- dent on DNA polymerase I without requiringany peculiarprop- sion of the polAl nonsense mutation. This mutation is readily erties of the DNA polymerase I. Other strains containing a ts suppressed by amber suppressors (11). In our spontaneous re- DNA polymerase III cannot replicate even ifthey contain DNA polymerase I. Table 1. Transduction of polA+ into HS432 Class of transductant Phenotype Number % Table 3. Loss of suppression is associated with loss of I -ValRMeMeSs, ts 35 54 temperature-resistant phenotype II Vals MeMeSs, ts 17 26 Infecting MeMeSR Temp. resistant m Val" MeMeS , 13 20 temp. resistant phage Strain Lysogens Cured Lysogens Cured ON1 (su+3) HM31 4/4 0/4 4/4 0/4 Recipient: HS432 (metE-,polAl,polBlOO,poIC1026, ValR, MeMeSs, ts). Donor: FTP439 (metE+, poLAU, poIC+, Vals, MeMeSR). Transduc- HM31 is an HS432 derivative containing lacam. Suppression in ly- tion was with initial selection forMet', followed by assessment of other sogens was scored by temperature-resistant, MeMeSR, and Lac' phe- markers. ValR is due to a mutation in the ilv genes (10). MeMeS re- notype. Selection for cured strains was by superinfection at high mul- sistance is conferred by DNA polymerase I (poU+). No MeMeSR, ts tiplicity of infection with 080, followed by 1-hr incubation at 3700 and transductants were found. Other classes arising are shown for com- overnight incubation at 32°C. Survivors were plated on EMB agar at parison. The order of relevant genes is: ilv (Val)-met-poLA. 32T and white (Lac-) colonies were analyzed. Downloaded by guest on September 27, 2021 7026 Genetics: Niwa et aL Proc. NatL Acad. Sci. USA 78 (1981)

Table 4. Transduction of poLA12 gene to temperature- Table 5. Linkage of TniO and pcbA resistant revertant Transductants Transductant Temp. Recipient class Phenotype Number % Recipient Donor TetR resistant % CSM61 (metEf) I Va1R MeMeSR, 75 50 E511 (polA,poICts) 61P-14 144 139 97 temp. resistant E486 (poLA+, polCts) 61P-14 220 195 89 II Vals MeMeSR, 45 30 temp. resistant TnlO was introduced into linkage with pcbA and the TetR marker m VaiR MeMeSR, ts 31 20 was transduced to recipient strains. The temperature-resistant phe- notype was scored by growth at 420C; TetR phenotype (the primary HS432 (metEf) I ValR MeMeSS, to 36 51 selection) was scored by growth on L-agar plates containing tetracy- cline at 25 II Vals MeMeSs, ts 12 17 pg/ml. m ValR MeMeSR, ts 22 32 the temperature-resistant phenotype caused by DNA poly- Donors: HS405 and 108 (poLA12, metE+). The metEf allele was trans- merase I. duced into CSM61 and HS432 by P1 from MJ245 to construct recipi- The Bypass Replication Pathway Depends on a Transduc- ents. DNA polymerase levels were measured to ensure that the metEf ible Locus and Is General. Our conclusion from the above re- transductanto remained polAl. MeMeS resistance was measured at 32°C. Selection was for MetE+, as in Table 1. sults is that HS432 contains a mutation that allows DNA rep- lication to proceed independent of DNA polymerase III but dependent on DNA polymerase I. A test ofthis conclusion was A ts DNA Polymerase I Prevents Temperature-Resistant to move this gene to another strain which contained polA+ and Phenotype. To test the conclusion that certain mutations in E. polCts and was ts. Such a test could also determine whether the coli allow replicative DNA synthesis that is dependent on DNA locus could suppress polCts alleles other than polC1026. We polymerase I, we used a ts DNA polymerase I mutation. Strains designate the putative locus allowing bypass ofdefects in poiC with the polA12 mutation do not have defective growth at a re- as pcbA (pol C bypass). We accomplished the goal of this test strictive temperature although they contain a DNA polymerase by using a transposon as a marker linked to pcbA. We linked I that has a ts synthesis activity when isolated. The lability of this transposon to pcbA as described and then used drug resis- the polymerase function in these mutants also was demon- tance as the selected marker in transduction to E511 and E486, strated by a lack of MalNEt-resistant synthesis in toluene- two polA' polCts strains that are ts. With high frequency (Table treated cells, such preparations behaving as Pol F. 5) the transductants showed conversion to temperature-resist- When the polA12 mutation was introduced into CSM61 [a ant phenotype. Thus, TnlO is closely linked to pcbA in strain spontaneous Pol I+ temperature-resistant revertant (5)] the 61P-14, and pcbA- can suppress the ts phenotype of polCts. transductants were ts again (Table 4). The DNA polymerase I This was true for at least three separate polCts alleles-polC1026 level was measured to verify the presence ofpolA12 in the class in HS432, polC511, and polC4%. III transductants. We conclude that DNA replication in the temperature-resistant mutation CSM61 is dependent on the DISCUSSION polymerase activity ofDNA polymerase I at 42°C and that when The evidence presented here shows that DNA replication can this activity is ts the strain becomes ts again. This is an important be dependent on DNA polymerase I synthesis activity and can observation because it also seems to rule out the possibility that proceed under conditions such that DNA polymerase III is in- a cryptic DNA polymerase is being activated in spontaneous active. DNA polymerase I synthesis activity is not normally re- revertants or that the introduction of a polA gene other than quired for DNA replication. The effect ofpcbA- is general and polAl activates the polC,, gene in a manner allowing temper- can phenotypically suppress different polCts alleles. Our con- ature-resistant phenotype. clusion is that E. coli has alternate pathways ofreplication. This Next we introduced polA12 into HS432 (Table 4). All Met' is reasonable in light of the evidence that DNA replication for transductants (receiving the polA region, in this case poLA12) certain bacteriophage can be initiated via several different remained ts, agreeing with the above results. Last, we intro- mechanisms (1). duced polC+ into representatives of this class (CSM61 polA12 Our evidence is that polC defects can be bypassed, depen- and HS432 polA12) by conjugation and recovered temperature- dent on the presence of DNA polymerase I and pcbA-. We do resistant recombinants. This argues that the polA12 gene does not know how replication is altered molecularly so that DNA not reverse the Pol III' phenotype. polymerase I can fulfill a critical role of DNA polymerase III. DNA Polymerase II Cannot Support Replication. We have It seems likely that the altered gene product involved can in- previously shown that we could not locate a mutation respon- teract with DNA polymerase I to complete a particular step. sible for the temperature-resistant phenotype near the polB lo- Given a primer, it is not unlikely that replication could proceed cus in the spontaneous revertants. We tested whether DNA by using DNA polymerase I activity alone. Thus, we might ex- polymerase II can support DNA replication, as DNA polymer- pect that the altered gene product can play a role in utilizing ase I can, in HS432. A wild-type polB gene was transduced into DNA polymerase I in primer synthesis. This would argue that an HS432 derivative, with leu used as a marker. We found that the alteration might lie in one of the several required all leu transductants remained ts (and also MeMeSs). In each for early priming steps or in the peptides found associated with transductant the presence of DNA polymerase I activity sec- the polC gene product to form holoenzyme. Elucidation ofthe ondary to introduction ofsu+3 in a phage lysogen caused a tem- alteration should clarify the role of DNA polymerase III in rep- perature-resistant phenotype. Of nine leu+ transductants lication, whether it is required for more than one step, and what screened for DNA polymerase II activity by partial enzyme properties allow it to be uniquely suited to the critical role. purification, four were polB+ as judged by enzyme levels, an Two points merit consideration. First, the replicative path- appropriate number for leulpolB linkage. We conclude that way demonstrated in our revertants is not aberrant but is au- DNA polymerase II cannot support replication in the HS432 thentic in that the dnaB gene is required. Thus, introduction background and that the wild-type polB allele does not prohibit ofdnaBts alleles makes the temperature-resistant revertants ts Downloaded by guest on September 27, 2021 Genetics: Niwa et aL Proc. NatL Acad. Sci. USA 78 (1981) 7027

(unpublished data). Second we do not know that the poiC pep- 4. Konrad, E. B. & Lehman, I. R. (1974) Proc. Natl Acad. Sci. USA tide is not needed as a stabilizing agent for some as yet unrec- 71, 2048-2051. ognized role in replication at the restrictive temperature, even 5. Niwa, O., Bryan, S. K. & Moses, R. E. (1979) Proc. NatL Acad. Sci. USA 76, 5572-5576. though the polymerase activity is not needed. 6. Niwa, O., Bryan, S. K. & Moses, R. E. (1980) Mechanistic Stud- ies ofDNA Replication and , ed. Alberts, This work was supported by U.S. Public Health Service Grant B. (Academic, New York). GM19122 and Robert A. Welch Foundation Grant Q-543. 7. Lennox, E. S. (1955) Virology 1, 190-206. 8. Bachman, B. & Low, K. B. (1980) Microbiol Rev. 44, 1-56. 1. Kornberg, A. (1980) DNA Replication (Freeman, San Francisco), 9. Kleckner, N., Roth, J. & Botstein, D. (1977) J. Mol Biol 116, pp. 347-414. 125-159. 2. Wickner, S. (1978) Annu. Rev. Biochem. 47, 1163-1191. 10. Murgola, E. J., Prather, N. E. & Hadley, K. H. (1978)J. Bacte- 3. Gefter, M. L., Hirota, Y., Kornberg, T., Wechsler, J. A. & Bar- riol 134, 801-07 noux, C. (1971) Proc. Natl Acad. Sci. USA 68, 3150-3153. 11. Gross, J. & Gross, M. (1969) Nature (London) 224, 1166-1168. Downloaded by guest on September 27, 2021