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DNA Polymerase I- Dependent Replication (Temperature-Sensitive Dna Mutants/Extragenic Suppression) OSAMI NIWA*, SHARON K

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DNA Polymerase I- Dependent Replication (Temperature-Sensitive Dna Mutants/Extragenic Suppression) OSAMI NIWA*, SHARON K Proc. Nati Acad. Sci. USA Vol. 78, No. 11, pp. 7024-7027, November 1981 Genetics Alternate pathways of DNA replication: DNA polymerase I- dependent replication (temperature-sensitive dna mutants/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 gene 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 Escherichia coli 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 polymerases 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' exonuclease 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 enzyme 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 genes 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 pyrophosphate 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 genome. 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. polymer- 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 Assay. 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
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