Proc. Natl. Acad. Sci. USA Vol. 84, pp. 4195-4199, June 1987 Genetics DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis (DNA replication/SOS repair) MICHAEL E. HAGENSEE, TERRY L. TIMME, SHARON K. BRYAN, AND ROBB E. MOSES Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 Communicated by Daniel Nathans, March 9, 1987 (receivedfor review January 7, 1987) ABSTRACT Strains of Escherichia coli possessing the polC+ by transduction. Strain ERli is an E486 (polC486) (8) pebAl mutation, a functional DNA polymerase I, and a derivative made by P1 transduction ofa TnJO linked topcbAl temperature-sensitive mutation in DNA polymerase m can from RM552. Strain RM552 is an ESli (8) derivative con- survive at the restrictive temperature (430C) for DNA poly- taining TnJO linked to pcbAl (zic-J: :TnJO) transduced from a merase m. The mutation rate of the bacterial genome of such CSM61 derivative (7). Strain SB229 was constructed by strains after exposure to either UV light or ethyl methanesul- transduction of recA56 srlJ300::TnJO into JM103. fonate was measured by its rifampicin resistance or amino acid Plasmid pDS4-26 was provided by C. McHenry. It contains requirements. In addition, Weigle mutagenesis of preirradi- the coding region for the a subunit of DNA polymerase III ated X phage was also measured. In all cases, no increase in (9). Plasmid pSB5 is a clone of the a subunit of DNA mutagenesis was noted at the restrictive temperature for DNA polymerase III derived in our laboratory. polymerase HI. Introduction of a cloned DNA polymerase HI Materials. Media were purchased from Difco. EtMes was gene returned the mutation rate of the bacterial genome as well obtained from Kodak, and rifampicin was purchased from as the Weigle mutagenesis to normal at 43C. Using a recA-LacZ Sigma. fusion, the SOS response after UV irradiation was measured Procedures. Phage X was grown and X lysogens were and found to be normal at the restrictive and permissive constructed using standard procedures. Transformation was temperature for DNA polymerase III, as was induction of X performed as described by Hanahan (10). prophage. Recombination was also normal at either tempera- To assess the mutation rate, cells were collected in ture. Our studies demonstrate that a functional DNA polymer- midlogarithmic phase and resuspended to half their original ase III is strictly required for mutagenesis at a step other than volume in 0.05 M K2HPO4 (pH 7.4). Cells were either SOS induction. UV-irradiated using a germicidal lamp (General Electric) with a UV flux of 1 J.m-2.sec-l at room temperature or The functional DNA polymerase III holoenzyme complex of exposed to EtMes at a concentration of 0.2 M at 32 or 43°C. Escherichia coli is composed ofat least seven proteins (1, 2). Samples were removed at time intervals to measure cell These components interact to replicate the E. coli genome at survival, concentrated, and plated on indicator plates at the high fidelity. The a subunit of this complex possesses the desired temperature (32 or 43°C). The indicator plates con- polymerizing activity and is encoded by the dnaE (polC) gene sisted of either L-agar with rifampicin at a concentration of (3). Temperature-sensitive mutations at this locus prevent 100 ,ug/ml or M9 plates lacking leucine. Mutation rate was replication at the restrictive temperature of 43°C (4). Muta- calculated as colonies on indicator plates divided by survi- tions at the dnaE locus may produce strains with an increase vors on L-agar. D37 values (the doses necessary to reduce the in the spontaneous mutation rate (5) as well as UV-induced surviving fractions to e-1 or 0.37) were determined graphi- mutagenesis (6). cally. The pcbAl mutation allows DNA polymerase I-dependent The Weigle mutagenesis experiments were performed replication ofthe bacterial genome without a functional DNA essentially as described by Defais et al. (11) using wild-type polymerase III a subunit (7). Strains containing the pcbAl X. Reactivation was determined from the total number of mutation, a functional DNA polymerase I, and a tempera- surviving plaques, whereas mutagenesis was determined ture-sensitive DNA polymerase III are viable at the restric- from the number of clear plaques. tive temperature (43°C) for DNA polymerase III and, there- The SOS response was measured by assaying ,B-galacto- fore, can be used to assess the role of DNA polymerase III sidase activity from a X lysogen carrying a recA-lacZ fusion in mutagenesis. Using such strains, we find that DNA (XGE190) provided by G. Weinstock (12). Units of *- polymerase III is required for mutagenesis of the bacterial galactosidase activity were as given by Miller (13). genome by either UV irradiation or ethyl methanesulfonate (EtMes) exposure as well as for Weigle mutagenesis. DNA RESULTS polymerase III is not required for induction of the SOS response or DNA recombination. Mutagenesis Requires DNA Polymerase III a-Subunit Ac- tivity. The strain CSM61 contains the pcbAl mutation, a functional DNA polymerase I, and a temperature-sensitive MATERIALS AND METHODS mutation in the a subunit of DNA polymerase III. DNA Strains. The bacterial strains used are listed in Table 1. replication in this strain at 43°C (7) requires DNA polymerase CSM61 and CSM14 are spontaneous, temperature-resistant I. CSM61 was exposed to UV irradiation, and rifampicin- revertants from HS432 (polAl, polB100, polC1O26, pcbAl) as resistant mutants arising were measured at 32 and 43°C. At previously described (7). CSM98 is a CSM61 derivative made the permissive temperature for DNA polymerase III (32°C), the number of mutations increased with increasing exposure to At the for DNA The publication costs of this article were defrayed in part by page charge UV light (Fig. 1B). restrictive temperature payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: EtMes, ethyl methanesulfonate. 4195 Downloaded by guest on September 29, 2021 4196 Genetics: Hagensee et al. Proc. Natl. Acad. Sci. USA 84 (1987) Table 1. Bacterial strains Relevant genotype or Strain phenotype Source 55. CSM14 polA+, polB100, polClO26, 50 pcbAl, Leu-, His-, tr This laboratory CSM61 polAl, polB100, polCJO26, pcbAl, sup(PolI), 45 Leu-, His-, tr This laboratory CSM98 polA+, polB100, polC+, 40 pcbAl, Leu-, His-, tr This laboratory 0) ER11 E486 (polC486), pcbAl ° 35 zic-l::TnlO, tr This laboratory SB229 JM103 recA56, 30 - srll300::TnlO This laboratory O 320C W3110 Wild type J. Cairns 0 25 - tr, Temperature-resistant. , 20 - polymerase III (43°C), the background spontaneous mutation rate is the same as at 32°C, but there was no increase in the 15- number of mutations with increasing exposure to UV light. CSM61 was also exposed to a second mutagen, EtMes, and 10 the results were similar (Fig. 1A). CSM61 is phenotypically PolI+ because of a suppressor polAl amber and in 5 430C mutation (7) acting on the mutation, 0 extracts this strain has 10-15% ofthe wild-type level of DNA I I I I I polymerase I activity. This level is sufficient to allow normal 0 20 40 60 80 100 120 140 DNA repair at 32°C and to support replication at 43°C. seconds of UV at 50cm However, one might question if the failure of mutagenesis at a level DNA I FIG. 2. UV mutagenesis in ERll. Mutants were scored as 43°C is due to low of polymerase activity. rifampicin-resistant colonies. D37 values were 35 sec at 32 and 43°C. Strain CSM14 is an isogenic strain with an intragenic muta- UV flux was 1 Jm-2-sec-1. tion producing a polA+ allele giving DNA polymerase I with normal properties after a 1000-fold purification (S.K.B. and R.E.M., unpublished results). Strain CSM14 shows the same These results were also confirmed by using a second response to UV irradiation (Fig. 1C) and EtMes exposure marker, leucine prototrophy, to score for mutations. Strains (not shown) as CSM61. We conclude that the lack of CSM61 and ERll showed less than 1 Leu+ revertant per 108 mutagenesis at 43°C is not due to low levels of DNA survivors at 430C after EtMes exposure for 30 min but polymerase I. A nonisogenic polCts strain containing pcbAl, showed greater than 20 Leu+ revertants per 108 survivors at ERll (polC486), also showed no increase in the UV-induced 320C. mutagenesis at the restrictive temperature (Fig. 2). The same To rule out a simple temperature effect on mutagenesis, we result occurred with EtMes. This indicates that the deficit at constructed a polC+ strain, CSM98, which is isogenic to 43°C is not polC allele-specific. CSM61 and CSM14. As seen in Fig. 3 A and B, this strain A B C 22 32°C 18 U) 0 :314 32°C Cu 0 10 20 30 0 40 80 120 0 20 60 100 140 minutes with .2M EMS seconds of UV at 50cm seconds of UV at 50cm FIG. 1. Mutagenesis in CSM61 after EtMes (EMS) (A) or UV (B) exposure and also in CSM14 after UV exposure (C). Mutants were scored as rifampicin-resistant colonies. D37 values at 32 and 43°C were 16 min in A and 32 sec in both B and C. UV flux was 1 J'm-2-sec-1. Cells from either strain held at 32 or 43°C in phosphate buffer did not show an increase in rifampicin-resistant mutants after 30 min. Survival rate was greater than 90%o in buffer at both temperatures. Downloaded by guest on September 29, 2021 Genetics: Hagensee et al.