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DNA Polymerase III from Escherichia coliCells Expressing mutA Mistranslator tRNA Is Error-prone

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m206856200

ISSN

1083-351X

Autores

Abu Amar M. Al Mamun, Kenneth J. Marians, M. Zafri Humayun,

Tópico(s)

CRISPR and Genetic Engineering

Resumo

Translational stress-induced mutagenesis (TSM) refers to the elevated mutagenesis observed in Escherichia coli cells in which mistranslation has been increased as a result of mutations in tRNA genes (such as mutA) or by exposure to streptomycin. TSM does not require lexA-regulated SOS functions but is suppressed in cells defective for homologous recombination genes. Crude cell-free extracts from TSM-induced E. coli strains express an error-prone DNA polymerase. To determine whether DNA polymerase III is involved in the TSM phenotype, we first asked if the phenotype is expressed in cells defective for all four of the non-replicative DNA polymerases, namely polymerase I, II, IV, and V. By using a colony papillation assay based on the reversion of alacZ mutant, we show that the TSM phenotype is expressed in such cells. Second, we asked if pol III from TSM-induced cells is error-prone. By purifying DNA polymerase III* from TSM-induced and control cells, and by testing its fidelity on templates bearing 3,N 4-ethenocytosine (a mutagenic DNA lesion), as well as on undamaged DNA templates, we show here that polymerase III* purified from mutA cells is error-prone as compared with that from control cells. These findings suggest that DNA polymerase III is modified in TSM-induced cells. Translational stress-induced mutagenesis (TSM) refers to the elevated mutagenesis observed in Escherichia coli cells in which mistranslation has been increased as a result of mutations in tRNA genes (such as mutA) or by exposure to streptomycin. TSM does not require lexA-regulated SOS functions but is suppressed in cells defective for homologous recombination genes. Crude cell-free extracts from TSM-induced E. coli strains express an error-prone DNA polymerase. To determine whether DNA polymerase III is involved in the TSM phenotype, we first asked if the phenotype is expressed in cells defective for all four of the non-replicative DNA polymerases, namely polymerase I, II, IV, and V. By using a colony papillation assay based on the reversion of alacZ mutant, we show that the TSM phenotype is expressed in such cells. Second, we asked if pol III from TSM-induced cells is error-prone. By purifying DNA polymerase III* from TSM-induced and control cells, and by testing its fidelity on templates bearing 3,N 4-ethenocytosine (a mutagenic DNA lesion), as well as on undamaged DNA templates, we show here that polymerase III* purified from mutA cells is error-prone as compared with that from control cells. These findings suggest that DNA polymerase III is modified in TSM-induced cells. Autonomous organisms normally replicate its DNA accurately, but the fidelity of replication can be transiently decreased in response to environmental and physiological stimuli through a number of pathways (1Drake J.W. Charlesworth B. Charlesworth D. Crow J.F. Genetics. 1998; 148: 1667-1686Crossref PubMed Google Scholar). Although the Escherichia coli SOS response represents the best-described transient mutator response (2Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 407-464Google Scholar), emerging evidence indicates the existence of multiple inducible mutagenic pathways inE. coli (3Humayun M.Z. Mol. Microbiol. 1998; 30: 905-910Crossref PubMed Scopus (72) Google Scholar). One especially intriguing pathway is provoked by increased translational errors resulting from mutations in tRNA genes (4Slupska M.M. Baikalov C. Lloyd R. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4380-4385Crossref PubMed Scopus (58) Google Scholar, 5Murphy H.S. Humayun M.Z. J. Bacteriol. 1997; 179: 7507-7514Crossref PubMed Google Scholar), in genes specifying tRNA-modification enzymes (6Zhao J. Leung H.E. Winkler M.E. J. Bacteriol. 2001; 183: 1796-1800Crossref PubMed Scopus (18) Google Scholar), or from exposure to streptomycin, an antibiotic that promotes mistranslation (7Balashov S. Humayun M.Z. J. Mol. Biol. 2002; 315: 513-527Crossref PubMed Scopus (40) Google Scholar). This pathway, dubbed translational stress-induced mutagenesis (TSM) 1The abbreviations used for: TSM, translational stress-induced mutagenesis; pol, polymerase; pol III HE, polymerase III holoenzyme; pol III*, polymerase III*; RF, replicative form II; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; IPTG, isopropyl-β-d-thiogalactopyranoside; nt, nucleotide; DTT, dithiothreitol; BSA, bovine serum albumin; SSB, single-stranded DNA-binding protein; ssDNA, single-stranded DNA; LM-PCR, ligation-mediated PCR. (3Humayun M.Z. Mol. Microbiol. 1998; 30: 905-910Crossref PubMed Scopus (72) Google Scholar), does not require the induction of lexA/recA-regulated SOS genes and is suppressed in cells defective for RecABC/RuvABC-dependent homologous recombination (5Murphy H.S. Humayun M.Z. J. Bacteriol. 1997; 179: 7507-7514Crossref PubMed Google Scholar, 7Balashov S. Humayun M.Z. J. Mol. Biol. 2002; 315: 513-527Crossref PubMed Scopus (40) Google Scholar, 8Ren L. Al Mamun A.A. Humayun M.Z. Mol. Microbiol. 1999; 32: 607-615Crossref PubMed Scopus (16) Google Scholar, 9Ren L. Mamun A.A. Humayun M.Z. J. Bacteriol. 2000; 182: 1427-1431Crossref PubMed Scopus (10) Google Scholar). Available genetic (5Murphy H.S. Humayun M.Z. J. Bacteriol. 1997; 179: 7507-7514Crossref PubMed Google Scholar, 7Balashov S. Humayun M.Z. J. Mol. Biol. 2002; 315: 513-527Crossref PubMed Scopus (40) Google Scholar, 8Ren L. Al Mamun A.A. Humayun M.Z. Mol. Microbiol. 1999; 32: 607-615Crossref PubMed Scopus (16) Google Scholar, 9Ren L. Mamun A.A. Humayun M.Z. J. Bacteriol. 2000; 182: 1427-1431Crossref PubMed Scopus (10) Google Scholar) and biochemical (10Al Mamun A.A. Rahman M.S. Humayun M.Z. Mol. Microbiol. 1999; 33: 732-740Crossref PubMed Scopus (20) Google Scholar) evidence suggests that the TSM phenotype results from error-prone DNA replication rather than from defective DNA repair. On the basis of the effect of elimination of individual genes encoding "non-replicative" polymerases, we previously proposed that either DNA polymerase III or an unidentified new DNA polymerase is responsible for error-prone replication in TSM-induced cells. DNA polymerase III holoenzyme (pol III HE) accounts for more than 90% of cellular DNA synthesis (11Kornberg A. Baker T. DNA Replication. W. H. Freeman & Co., New York1991: 165-194Google Scholar, 12Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar) and is also required for the major post-replicative mismatch correction pathway (13Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (776) Google Scholar, 14Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar). pol III HE was shown to effectively carry out translesion DNA synthesis past abasic sites, mostly producing −1-bp deletions (15Tomer G. Livneh Z. Biochemistry. 1999; 38: 5948-5958Crossref PubMed Scopus (29) Google Scholar), as contrasted to translesion synthesis carried out by pol V, which mostly yields base substitutions at abasic sites. pol III HE is a 10-subunit polymerase ((αεθ)2τ2γ1δδ′χγ(β2)2) consisting of three main components (16Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 17Pritchard A.E. Dallmann H.G. Glover B.P. McHenry C.S. EMBO J. 2000; 19: 6536-6545Crossref PubMed Google Scholar) as follows: 1) the core polymerase (αεθ), responsible for DNA synthesis and proofreading (18McHenry C.S. Crow W. J. Biol. Chem. 1979; 254: 1748-1753Abstract Full Text PDF PubMed Google Scholar, 19Scheuermann R. Tam S. Burgers P.M. Lu C. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7085-7089Crossref PubMed Scopus (146) Google Scholar); 2) the processivity factor or β-sliding clamp (β2) (20LaDuca R.J. Crute J.J. McHenry C.S. Bambara R.A. J. Biol. Chem. 1986; 261: 7550-7557Abstract Full Text PDF PubMed Google Scholar) that tethers the polymerase to the DNA (21Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar); and 3) the DnaX complex, containing δδ′, χ, ψ, and either or both of two different DnaX proteins (γ and τ), that is responsible for loading the β-processivity clamp onto the DNA (22McHenry C. Kornberg A. J. Biol. Chem. 1977; 252: 6478-6484Abstract Full Text PDF PubMed Google Scholar, 23Fay P.J. Johanson K.O. McHenry C.S. Bambara R.A. J. Biol. Chem. 1982; 257: 5692-5699Abstract Full Text PDF PubMed Google Scholar). The largest subassembly of pol III HE is DNA polymerase III* (pol III*) composed of all the subunits of HE except the β-subunit ((αεθ)2τ2γ1δδ′χγ) (24Wickner W. Schekman R. Geider K. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1764-1767Crossref PubMed Scopus (70) Google Scholar). The HE is completed by the addition of (β2)2 to pol III* (22McHenry C. Kornberg A. J. Biol. Chem. 1977; 252: 6478-6484Abstract Full Text PDF PubMed Google Scholar, 24Wickner W. Schekman R. Geider K. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1764-1767Crossref PubMed Scopus (70) Google Scholar). In addition to pol III, four additional DNA polymerases (polymerases I, II, IV, and V) are known in E. coli. Even though these polymerases carry out important cellular functions, genes encoding each of these polymerases can be mutationally inactivated, implying that considerable functional redundancy is built into the replication apparatus. DNA polymerase I (pol I; encoded by polA), known as a "repair polymerase," normally functions to fill gaps that arise during lagging strand replication and during excision repair. The remaining three DNA polymerases, namely II (pol II; encoded bypolB (25Bonner C.A. Hays S. McEntee K. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7663-7667Crossref PubMed Scopus (102) Google Scholar, 26Chen H. Sun Y. Stark T. Beattie W. Moses R.E. DNA Cell Biol. 1990; 9: 631-635Crossref PubMed Scopus (12) Google Scholar)), IV (pol IV; encoded by dinB(27Wagner J. Gruz P. Kim S.R. Yamada M. Matsui K. Fuchs R.P. Nohmi T. Mol. Cell. 1999; 4: 281-286Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar)), and V (pol V; encoded by umuDC (28Reuven N.B. Arad G. Maor-Shoshani A. Livneh Z. J. Biol. Chem. 1999; 274: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 29Tang M. Shen X. Frank E.G. O'Donnell M. Woodgate R. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8919-8924Crossref PubMed Scopus (488) Google Scholar)), are induced as a part of the SOS system. pol II has been proposed to play a role in replication-restart following DNA damage, a process that bypasses DNA damage in both an error-free (30Rangarajan S. Woodgate R. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9224-9229Crossref PubMed Scopus (131) Google Scholar, 31Pham P. Rangarajan S. Woodgate R. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8350-8354Crossref PubMed Scopus (79) Google Scholar) and error-prone manner (32Napolitano R. Janel-Bintz R. Wagner J. Fuchs R.P. EMBO J. 2000; 19: 6259-6265Crossref PubMed Scopus (330) Google Scholar, 33Becherel O.J. Fuchs R.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8566-8571Crossref PubMed Scopus (84) Google Scholar). pol IV is involved in certain types of untargeted SOS mutagenesis (27Wagner J. Gruz P. Kim S.R. Yamada M. Matsui K. Fuchs R.P. Nohmi T. Mol. Cell. 1999; 4: 281-286Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar,34Brotcorne-Lannoye A. Maenhaut-Michel G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3904-3908Crossref PubMed Scopus (117) Google Scholar, 35Kim S.R. Maenhaut-Michel G. Yamada M. Yamamoto Y. Matsui K. Sofuni T. Nohmi T. Ohmori H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13792-13797Crossref PubMed Scopus (295) Google Scholar), whereas pol V, working in conjunction with a number of other factors including the RecA protein, is believed to be responsible for translesion DNA synthesis (28Reuven N.B. Arad G. Maor-Shoshani A. Livneh Z. J. Biol. Chem. 1999; 274: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 29Tang M. Shen X. Frank E.G. O'Donnell M. Woodgate R. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8919-8924Crossref PubMed Scopus (488) Google Scholar, 36Pham P. Bertram J.G. O'Donnell M. Woodgate R. Goodman M.F. Nature. 2001; 409: 366-370Crossref PubMed Scopus (110) Google Scholar). However, pol V may also be responsible for untargeted mutations at undamaged template sites (37Fijalkowska I.J. Dunn R.L. Schaaper R.M. J. Bacteriol. 1997; 179: 7435-7445Crossref PubMed Google Scholar,38Maor-Shoshani A. Reuven N.B. Tomer G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 565-570Crossref PubMed Scopus (93) Google Scholar). The individual loss of polA, polB,dinB, or umuDC genes does not affect the expression of the TSM response (5Murphy H.S. Humayun M.Z. J. Bacteriol. 1997; 179: 7507-7514Crossref PubMed Google Scholar, 8Ren L. Al Mamun A.A. Humayun M.Z. Mol. Microbiol. 1999; 32: 607-615Crossref PubMed Scopus (16) Google Scholar, 10Al Mamun A.A. Rahman M.S. Humayun M.Z. Mol. Microbiol. 1999; 33: 732-740Crossref PubMed Scopus (20) Google Scholar). However, an analysis based on cells with defects in individual genes leaves open the possibility that two or more of these four nonessential DNA polymerases may have nonexclusive (redundant) roles in error-prone DNA synthesis. Here we show that a strain simultaneously defective for pol I, pol II, pol IV, and pol V can be constructed, proving that loss of all four non-replicative polymerases is compatible with viability. Analysis of the TSM response in this strain confirms that the four non-replicative polymerases are not required collectively or individually for the TSM response. To address the question directly whether pol III HE or an unknown 6th DNA polymerase is responsible for mistranslation-induced mutagenesis, we purified pol III* from TSM-induced and uninduced cells and analyzed its replication fidelity on both damaged and undamaged template DNA. Our data show that purified pol III* from TSM-induced cells shows elevated mutagenesis on undamaged DNA as well as at a site-specific mutagenic lesion. TaqDNA polymerase was from Roche Molecular Biochemicals; restriction endonuclease BglI, T4 DNA ligase, Vent DNA polymerase, and T4 polynucleotide kinase were from New England Biolabs. The β subunit was purified as described by Johanson et al. (39Johanson K.O. Haynes T.E. McHenry C.S. J. Biol. Chem. 1986; 261: 11460-11465Abstract Full Text PDF PubMed Google Scholar). The primase DnaG was purified as described by Marians (40Marians K.J. Methods Enzymol. 1995; 262: 507-521Crossref PubMed Scopus (54) Google Scholar). The E. coli single-stranded DNA-binding protein (SSB) was purified according to Minden and Marians (41Minden J.S. Marians K.J. J. Biol. Chem. 1985; 260: 9316-9325Abstract Full Text PDF PubMed Google Scholar). Plasmid pMV22 was constructed from plasmid pHM22 (5Murphy H.S. Humayun M.Z. J. Bacteriol. 1997; 179: 7507-7514Crossref PubMed Google Scholar) as follows. TheSphI-HindIII 1604-bp fragment containing thelacI q gene and the wild type glyVtRNA gene under Ptrc promoter was inserted into the low copy number plasmid pMW119 (42Al Mamun A.A. Tominaga A. Enomoto M. J. Bacteriol. 1996; 178: 3722-3726Crossref PubMed Google Scholar) cut with SphI andHindIII. Similarly, plasmid pMV11 was constructed by inserting the SphI-HindIII 1604-bp fragment containing the mutant glyV tRNA gene (mutA) from pHM11 (5Murphy H.S. Humayun M.Z. J. Bacteriol. 1997; 179: 7507-7514Crossref PubMed Google Scholar) into the SphI-HindIII sites of pMW119. To enable the subsequent transduction of the polA1 allele in strain HSM83 (see Table I for genotypes), the metE::Tn10 marker from strain CAG18491 (43Singer M. Baker T.A. Schnitzler G. Deischel S.M. Goel M. Dove W. Jaacks K.J. Grossman A.D. Erickson J.W. Gross C.A. Microbiol. Rev. 1989; 53: 1-24Crossref PubMed Google Scholar) was first transferred to HMS83 (44Campbell J.L. Shizuya H. Richardson C.C. J. Bacteriol. 1974; 119: 494-499Crossref PubMed Google Scholar) by P1 transduction. The transductants were selected on LB agar containing 30 μg/ml tetracycline (Sigma). One isolate, named AM132, was characterized by its inability to grow in minimal medium without methionine. E. coli strains AM134 and AM135 were constructed by co-transducing the polA1 allele along with the markermetE::Tn10 from strain AM132 into strains CC105 and CC105mutA, respectively. Strains AM134 and AM135 were characterized for the presence of polA1 allele by testing their sensitivity to 0.04% methyl methanesulfonate in LB plates and to UV irradiation at 7 J/m2 as described previously (45De Lucia P. Cairns J. Nature. 1969; 224: 1164-1166Crossref PubMed Scopus (430) Google Scholar). The E. coli strain AM130 was created by P1 transduction of the Δ(umuDC)595::Cmr allele from strain RW82 to strain AM107 (CC105 polB (10Al Mamun A.A. Rahman M.S. Humayun M.Z. Mol. Microbiol. 1999; 33: 732-740Crossref PubMed Scopus (20) Google Scholar)). The presence of the Δ(umuDC)595 allele was confirmed by UV sensitivity at 30 J/m2. The E. coli strain AM146 was constructed by transferring the ΔdinB::Kanr allele from strain YG7207 to strain AM130 by P1 transduction. The presence of the ΔdinB::Kanr allele in AM146 was confirmed by PCR amplification (forward primer 5′-CGCTGTATCAATACTTTGGTCA; reverse primer 5′-AGGCGAATAAGTTTTGTTTTGA) followed by analysis of restriction digestion patterns of the PCR products. The E. coli strain AM147 was made by co-transducing the polA1 allele along with the marker metE::Tn10 from strain AM132 to AM146 by P1 transduction. Strain AM147 was characterized by sensitivity to 0.04% methyl methanesulfonate and to UV irradiation at 7 J/m2.Table IBacterial and plasmid strainsStrainRelevant genotypeSource (Ref.)A. E. coliAM107araΔ(lac-proB) xiii polBΔ1∷Ω Sm-Sp Smr F′lacIZ proB +This laboratory (10Al Mamun A.A. Rahman M.S. Humayun M.Z. Mol. Microbiol. 1999; 33: 732-740Crossref PubMed Scopus (20) Google Scholar)AM130Δ(umuDC)595∷Cmr from RW82 in AM107This studyAM132metE0-3079∷Tn10(Tetr) from CAG18491 in HMS83This studyAM134polA1(Am),metE0-3079∷Tn10(Tetr) from AM132 in CC105This studyAM135polA1(Am),metE-3079∷Tn10 (Tetr) from AM132 in CC105mutAThis studyAM146ΔdinB∷Kanr from YG7207 in AM130This studyAM147polA1(Am),metE0-3079∷Tn10 (Tetr) from AM132 in AM146This studyAM155ΔdinB∷Kanr from YG7207 in CC104This studyCAG18491λ-, rph-1, metE0-3079∷Tn10 (Tetr)M. Berlyn (43Singer M. Baker T.A. Schnitzler G. Deischel S.M. Goel M. Dove W. Jaacks K.J. Grossman A.D. Erickson J.W. Gross C.A. Microbiol. Rev. 1989; 53: 1-24Crossref PubMed Google Scholar)CC104ara Δ(lac-proB) xiii F′lacIZ proB +J. Miller (69Nghiem Y. Cabrera M. Cupples C.G. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2709-2713Crossref PubMed Scopus (297) Google Scholar, 70Cupples C.G. Cabrera M. Cruz C. Miller J.H. Genetics. 1990; 125: 275-280Crossref PubMed Google Scholar)CC105ara Δ(lac-proB) xiiiF′lacIZ proB +J. Miller (69Nghiem Y. Cabrera M. Cupples C.G. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2709-2713Crossref PubMed Scopus (297) Google Scholar, 70Cupples C.G. Cabrera M. Cruz C. Miller J.H. Genetics. 1990; 125: 275-280Crossref PubMed Google Scholar)CC105mutAmutA590C in CC105J. Miller (58Michaels M.L. Cruz C. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9211-9215Crossref PubMed Scopus (40) Google Scholar)HMS83polB100, lacz53 (Am), λ−, thyA36,IN(rrnD-rrnE)1, psL151Smr, polA1(Am), rha-5, deoC2?, lys-34M. Berlyn (44Campbell J.L. Shizuya H. Richardson C.C. J. Bacteriol. 1974; 119: 494-499Crossref PubMed Google Scholar)MC1061hsdR hsdM+ araD Δ(ara-leu) Δ(lacIPOZY) galU galK strAR. M. Schaaper (71Casadaban M.J. Cohen S.N. J. Mol. Biol. 1980; 138: 179-207Crossref PubMed Scopus (1753) Google Scholar)NR9099ara Δ(pro-lac) recA56 thiF′(proAB lacIq ZΔM15)R. M. Schaaper (60Schaaper R.M. Danforth B.N. Glickman B.W. Gene (Amst.). 1985; 39: 181-189Crossref PubMed Scopus (95) Google Scholar)RW82Δ(umuDC)595∷Cmr uvrA6R. Woodgate (72Woodgate R. Mutat. Res. 1992; 281: 221-225Crossref PubMed Scopus (98) Google Scholar)YG7207ΔdinB∷Kanr in AB1157T. Nohmi (35Kim S.R. Maenhaut-Michel G. Yamada M. Yamamoto Y. Matsui K. Sofuni T. Nohmi T. Ohmori H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13792-13797Crossref PubMed Scopus (295) Google Scholar)B. PlasmidspHM11pSE380 with mutant glycine tRNAThis lab (5Murphy H.S. Humayun M.Z. J. Bacteriol. 1997; 179: 7507-7514Crossref PubMed Google Scholar)pHM22pSE380 with wild-type glycine tRNAThis lab (5Murphy H.S. Humayun M.Z. J. Bacteriol. 1997; 179: 7507-7514Crossref PubMed Google Scholar)pMV11Mutant glycine tRNA withlacI q in pMW119 from pHM11This studypMV22Wild-type glycine tRNA with lacI q in pMW119 from pHM22This studypMW119Ampr low-copy number (5–6 copies/cell) plasmid vectorA. A. M. Al Mamun (42Al Mamun A.A. Tominaga A. Enomoto M. J. Bacteriol. 1996; 178: 3722-3726Crossref PubMed Google Scholar)pSE380Ampr vectorR. Maurer (73Brosius J. DNA (New York). 1989; 8: 759-777Crossref PubMed Scopus (126) Google Scholar) Open table in a new tab The F′ factors contained in strains CC104 and CC107 (two strains related to CC105, the parental strain for AM147) contain a second copy of the dinB gene (46Kim S.R. Matsui K. Yamada M. Gruz P. Nohmi T. Mol. Genet. Genomics. 2001; 266: 207-215Crossref PubMed Scopus (166) Google Scholar, 47Strauss B.S. Roberts R. Francis L. Pouryazdanparast P. J. Bacteriol. 2000; 182: 6742-6750Crossref PubMed Scopus (74) Google Scholar). To show that AM147 does not have a second (wild type) copy of dinB, we carried out a PCR-based analysis as summarized in Fig.1. This analysis shows that an "external" primer set (F1/R1 in Fig. 1) yields a single band corresponding to a disrupted version of dinB in AM147 cells (lane 2) and that two "internal" primer sets (F2/R2 and F3/R3) do not yield a band (lanes 4 and6), indicating the loss of sequences corresponding todinB. To verify that we could have detected a second copy ofdinB, we repeated the analysis with AM155 (CC104 ΔdinB::Kanr), a strain harboring an F′ factor that was previously shown to have a second, episomal copy ofdinB. Our analysis shows that the F1/R1 primer set yields two bands (lane 8), one corresponding to a disrupted allele (1862 bp) and the other corresponding to the wild type allele (1603 bp). As expected, internal primer sets F2/R2 and F3/R3 amplify the second copy of dinB in AM155 cells. Thus, these data confirm that AM147 does not have an undisrupted dinB allele. Papillation assays were performed with slight modifications of the procedures described by Miller (48Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). Cultures were spread on minimal A medium containing 0.2% glucose, 500 μg/ml phenyl-β-d-galactopyranoside (a non-inducing lactose analog that serves as a carbon source after exhaustion of glucose; Sigma), 40 μg/ml of the β-galactosidase indicator 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal; Gold Biotechnologies), and 1 mmisopropyl-β-d-thiogalactopyranoside (IPTG; Sigma). Plates were incubated at 37 °C for 4–5 days at which time blue papillae (lacZ + revertants) are distinctly visible against the white background of lacZ −cells. M13 single-stranded DNA (ssDNA) molecules bearing an εC residue were constructed following the procedures described elsewhere (49Palejwala V.A. Pandya G.A. Bhanot O.S. Solomon J.J. Murphy H.S. Dunman P.M. Humayun M.Z. J. Biol. Chem. 1994; 269: 27433-27440Abstract Full Text PDF PubMed Google Scholar, 50Palejwala V.A. Wang G.E. Murphy H.S. Humayun M.Z. J. Bacteriol. 1995; 177: 6041-6048Crossref PubMed Google Scholar, 51Rahman M.S. Dunman P.M. Wang G. Murphy H.S. Humayun M.Z. Mol. Microbiol. 1996; 22: 747-755Crossref PubMed Scopus (10) Google Scholar) and summarized below. M13mp7L2 ssDNA was linearized by cutting with the restriction endonuclease EcoRI that cuts a hairpin DNA structure within the polycloning site of the vector. The linearized DNA was annealed to a 57-nt "scaffold" and a 5′-phosphorylated 17-nt insert containing a single site-specific εC lesion. Annealing of the 57-mer draws the two ends of the linear M13 ssDNA together to form a non-covalently closed circular DNA with a 17-nt "gap" complementary to the lesion-containing 17-mer. The annealed DNA is subjected to DNA ligation to generate a covalently closed ssDNA circle containing the lesion-bearing 17-nt insert. After the ligation step, the scaffold is removed by heat denaturation in the presence of a 10-fold molar excess of an "anti-scaffold" 57-mer (i.e. a 57-mer with a sequence complementary to that of the scaffold). The constructed circular ssDNA was purified by using a QIAquick gel extraction kit column (Qiagen). To prime in vitro DNA synthesis, a 60-mer was annealed to the ssDNA (5′-TAACCAATAGGAACGCCATCAAAAATAATTCGCGTCTGGCCTTCCTGTAGCCAGCTTTCA-3′; complementary to the M13 sequence from 6628 to 6687; Fig.4 A) as follows. One pmol of the ssDNA construct (with or without an εC lesion) was mixed with 1.5 pmol of a 60-mer in 50 mm Tris-Cl (pH 7.8), 10 mm MgCl2, and 20 mm dithiothreitol (DTT), heated at 85 °C for 5 min, and allowed to cool over 2 h to 30 °C. The terms εC-ssDNA and C-ssDNA, respectively, refer to the primed ssDNA bearing a site-specific εC lesion or normal cytosine (as a control). Buffers contain the following: "Tris-back extraction" buffer (TBEB): 50 mm Tris-Cl (pH 7.5), 100 mm NaCl, 1 mm EDTA, 20% glycerol, and 5 mm DTT; "0.20 TBEB" is TBEB containing 0.2 g/ml of ammonium sulfate; "0.17 TBEB" is TBEB containing 0.17 g/ml of ammonium sulfate (52Cull M.G. McHenry C.S. Methods Enzymol. 1995; 262: 22-35Crossref PubMed Scopus (41) Google Scholar). Buffer A is 50 mm Tris-Cl (pH 7.5), 0.5 mm EDTA, 20% glycerol, and 5 mm DTT. Buffer B is buffer A supplemented with 5 mm MgCl2, 0.2 mm EDTA and 0.4 mm ATP. pol III* was purified following the procedures described previously (23Fay P.J. Johanson K.O. McHenry C.S. Bambara R.A. J. Biol. Chem. 1982; 257: 5692-5699Abstract Full Text PDF PubMed Google Scholar), as summarized below. Cells were grown in 300 liters of LB medium supplemented with ampicillin (50 μg/ml) and IPTG (1 mm) as appropriate, and cells were harvested by centrifugation at 4 °C in a Sharples centrifuge. The cell pellet was resuspended in an equal volume of 50 mmTris-Cl (pH 7.5), 10% sucrose solution, quick-frozen in liquid nitrogen, and stored at −80 °C. The cell suspension (2 kg) was thawed on ice, and theA 595 was adjusted to 200 with 50 mm Tris-Cl (pH 7.5), 10% sucrose solution. One-ninth volume of lysis buffer (1.5 m NaCl, 0.2 m EDTA, 0.2 m spermidine, and 50 mm DTT) was added, and the pH was adjusted to 8.5 by adding solid Tris base. Lysozyme (10 mg/ml in water) was added to 0.2 mg/ml, and the contents were incubated for 30 min on ice, followed by 10 min in a 37 °C water bath. The lysed cells were distributed into 250-ml Sorvall GSA bottles, kept on ice for 5 min, and centrifuged for 60 min at 13,000 rpm in a Sorvall SLA-1500 rotor. The supernatant (fraction 1a) was collected by gentle decantation. To precipitate nucleic acid, polymin P (1% v/v; AmershamBiosciences) was added slowly, with gentle stirring, to fraction 1a to a final concentration of 0.06%. Stirring was continued for an additional 30 min at 4 °C. The suspension was centrifuged at 13,000 rpm for 30 min at 4 °C in a Sorvall SLA-1500 rotor to collect the supernatant (fraction 1b). Ammonium sulfate (0.25 g/ml) was added to fraction 1b over a 10-min interval while stirring. The contents were stirred for an additional 30 min at 4 °C, transferred into Sorvall GSA bottles, and centrifuged at 27,000 × g in a Sorvall SLA-1500 rotor for 45 min at 4 °C. The precipitate was resuspended in 0.2 TBEB (1/10th of the fraction 1a volume), and the insoluble fraction was collected by centrifugation as above. This procedure was repeated with 0.17 TBEB (1/40th of the fraction 1a volume), and the final recovered insoluble fraction was dissolved in a small volume of TBEB (fraction II). Fraction II was quick-frozen in liquid nitrogen and stored at −80 °C. Fraction II was thawed and dialyzed overnight against 4 liters of buffer A + 40 mm NaCl at 4 °C. The dialysate was clarified by centrifugation for 20 min at 48,200 × gat 4 °C. The clarified solution was diluted with buffer A to the conductivity of buffer A + 50 mm NaCl. The diluted fraction II was applied to a heparin-agarose (Sigma) column (1 ml of resin per 10 mg of protein) equilibrated with buffer A + 50 mm NaCl. The column was washed with 2 column volumes of the equilibration buffer, and the activity was eluted with a 10-column volume of NaCl gradient (50–400 mm) in buffer A. Fractions were pooled, and ammonium sulfate was added to 0.26 g/ml. The suspension was stirred for 2 h at 4 °C and centrifuged at 37,000 rpm for 60 min at 4 °C in a Sorvall A841 rotor. The precipitate was resuspended in a small volume (300 μl) of buffer B (fraction III). Fraction III was clarified by centrifugation for 5 min at 13,000 rpm in a microcentrifuge. The clarified fraction III was gel-filtered through a fast protein liquid chromatography Superose 6 column (HR 10/30;Amersham Biosciences) equilibrated with buffer B at a flow rate of 0.1 ml/min. Peak fractions were pooled, and glycerol was added to achieve a final concentration of 38% (fraction IV) and stored in aliquots at −80 °C. The assay mix (25 μl) containing 50 mm HEPES-KOH (pH 8.0), 50 mm potassium glutamate, 10 mm magnesium acetate, 10 mm DTT, 10 μg/ml rifampicin, 100 μg/ml bovine serum albumin (BSA), 24 μg/ml SSB, 3.2 μg/ml M13 Gori ssDNA, 100 μm each of CTP, GTP, and UTP, 8

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