Abstract
An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective s subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122:: Tn 5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC + plasmid. Interestingly, while the frequency of UV-induced GC → AT transitions is greatly reduced, the frequency of AT → TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC +; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.
Similar content being viewed by others
References
Bailone A, Sommer S, Knezevic J, Dutreix M, Devoret R (1991) A RecA protein mutant deficient in its interaction with the UmuDC complex. Biochimie 73:479–484
Banerjee SK, Borden A, Christensen RB, LeClerk JE, Lawrence CW (1990) SOS-dependent replication pasta single trans — syn cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells. J Bacteriol 172:2105–2112
Bridges BA, Woodgate R (1984) Mutagenic repair in Escherichia coli. The umuC gene product may be required for replication past pyrimidine dimers but not for the coding error in UV mutagenesis. Mol Gen Genet 196:364–366
Bridges BA, Woodgate R (1985) Mutagenic repair in Escherichia coli. Products of the recA gene and of the umuD and umuC genes act at different steps in UV-induced mutagenesis. Proc Natl Acad Sci USA 82:4193–4197
Bridges BA, Mottershead RP, Sedgwick SG (1976) Mutagenic repair in Escherichia coli. Requirement for a function of DNA polymerase III in ultraviolet light mutagenesis. Mol Gen Genet 144:53–58
Burckhardt SE, Woodgate R, Scheuermann RH, Echols H (1988) The UmuD mutagenesis protein of Escherichia coli: overproduction, purification and cleavage by RecA. Proc Natl Acad Sci USA 85:1811–1815
Ciesla Z (1982) Plasmid pKM101 mediated mutagenesis in Escherichia coli is inducible. Mol Gen Genet 186:298–300
Ciesla Z, O'Brien P, Clark AJ (1987) Genetic analysis of UV mutagenesis of the Escherichia coli glyU gene. Mol Gen Genet 207:1–8
Cox EC, Horner DI (1982) Dominant mutators in Escherichia coli. Genetics 100:7–18
Dengen GE, Cox EC (1974) Conditional mutator gene in Escherichia coli: isolation, mapping and effector studies. J Bacteriol 117:477–487
Donelly CE, Walker CG (1989) groE mutants of Escherichia coli are defective in umuDC dependent UV mutagenesis. J Bacteriol 181:6117–6125
Echols H, Goodman MF (1990) Mutation induced by DNA damage: a many protein affair. Mutat Res 236:301–311
Elledge SJ, Walker GC (1983) Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli. J Mol Biol 164:175–192
Foster PL, Sullivan AD (1988) Interaction between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli Mol Gen Genet 214:467–471
Fox TD, Weiss-Brummer B (1980) Leaky +1 and −1 frameshift mutations at the same site in a yeast mitochondrial gene. Nature 288:60–63
Frank EG, Hauser J, Levine AS, Woodgate R (1993) Targeting of the UmuD, UmuD′ and MucA′ mutagenesis proteins to DNA by RecA protein. Proc Natl Acad Sci USA 90:8169–8173
Hagensee ME, Timme T, Bryan SK, Moses RE (1987) DNA polymerase III of Escherichia coli is required for UV and ethyl methansulfonate mutagenesis. Proc Natl Acad Sci USA 84:4195–4199
Horiuchi T, Maki H, Sekiguchi M (1978) A new conditional lethal mutator dnaQ49 in Escherichia coli. Mol Gen Genet 163:277–283
Jonczyk P, Fijalkowska I, Ciesla Z (1988) Overproduction of the ε subunit of DNA polymerase III counteracts the SOS mutagenic response of Escherichia coli. Proc Natl Acad Sci USA 85:9124–9127
Kato T, Shinoura Y (1977) Isolation and characterization of mutants of Esherichia coli deficient in the induction of mutations by ultraviolet light. Mol Gen Genet 156:121–131
Kornberg A (1980) DNA replication. Freeman, San Francisco
Lawrence CW, Gibbs PEM, Borden A, Horsfall MJ, Kilbey BJ (1993) Mutagenesis induced by single UV photoproducts in E. coli and yeast. Mutat Res 299:157–163
Little JW (1983) The SOS regulatory system: control of its state by the level of RecA protease. J Mol Biol 167:791–808
Little JW, Mount DW (1982) The SOS regulatory system of E. coli. Cell 29:11–22
Maki H, Kornberg A (1987) Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits. Proc Natl Acad Sci USA 84:4389–4392
Marsh L, Walker GC (1985) Cold sensitivity induced by overproduction of UmuDC in Escherichia coli. J Bacteriol 162:155–161
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Miller JH, Low KB (1984) Specificity of mutagenesis resulting from the induction of the SOS system in the absence of mutagenic treatment. Cell 37:675–682
Minton NP (1984) Improved plasmid vectors for the isolation of translational lac fusions. Gene 31:269–273
Nohmi T, Battista JR, Dodson LA, Walker GC (1988) RecA-mediated cleavage activates UmuD for mutagenesis:mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci USA 85:1816–1820
Perry KL, Elledge SJ, Mitchell BB, Marsh L, Walker GC (1985) umuDC and mucAB operons whose products are required for UV light and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology. Proc Natl Acad Sci USA 82:4331–4335
Rajagopalan M, Lu CZ, Woodgate R, O'Donnell M, Goodman MF, Echols H (1992) Activity of the purified mutagenesis proteins UmuC, UmuD′ and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III. Proc Natl Acad Sci USA: 89:10777–10781
Sargentini NJ, Smith KC (1989) Mutational spectrum analysis of umuC-independent and umuC-dependent γ-radiation mutagenesis in Escherichia coli. Mutat Res 211:193–203
Scheuermann RH, Echols H (1984) A separate editing exonuclease for DNA replication. The ε subunit of Escherichia coli DNA polymerase III. Proc Natl Acad Sci USA 81:7747–7751
Shinagawa H, Kato T, Ise T, Makino K, Nakata A (1983) Cloning and characterization of the umu operon responsible for the inducible mutagenesis in Escherichia coli. Gene 23:167–174
Shinagawa H, Iwasaki H, Kato T, Nakata A (1988) RecA protein dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci USA 85:1806–1810
Sledziewska-Gójska E, Janion C (1989) Alternative pathways of methyl methansulfonate induced mutagenesis in Escherichia coli. Mol Gen Genet 216:126–131
Sledziewska-Gójska E, Grzesiuk E, Plachta A, Janion C (1992) Mutagenesis of Escherichia coli: a method for determining mutagenic specificity by analysis of tRNA suppressors. Mutagenesis 7:41–46
Sommer S, Knezevic J, Bailone A, Devoret R (1993) Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli. Mol Gen Genet 239:137–144
Steiborn G (1978) Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis. Isolation of uvm mutants and their phenotypic characterization in DNA repair and mutagenesis. Mol Gen Genet 165:87–93
Studwell PS, O'Donnell M (1990) Processive replication is contingent on the exonuclease subunit of DNA polymerase III holoenzyme. J Biol Chem 265:1171–1178
Sweasy JB, Witkin EM, Sinha N, Roegner-Maniscalco V (1990) RecA protein of Escherichia coli has a third essential role in SOS mutator activity. J Bacteriol 172:3030–3036
Takano K, Nakabeppu Y, Maki H, Horiuchi T, Sekiguchi M (1986) Structure and function of dnaQ and mutD mutators of Escherichia coli. Mol Gen Genet 205:9–13
Tessman I, Liu SK, Kennedy MA (1992) Mechanism of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine. Proc Natl Acad Sci USA 89:1159–1163
Walker GC (1984) Mutagenesis and inducible responses to DNA damage in Escherichia coli. Microbiol Rev 48:60–93
Witkin EM (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 40:869–907
Witkin EM, Kogoma T (1984) Involvement of the activated form of RecA protein in SOS mutagenesis and stable DNA replication in Escherichia coli. Proc Natl Acad Sci USA 81:7539–7543
Woodgate R (1992) Construction of a umuDC operon substitution mutation in Escherichia coli. Mutat Res 281:221–225
Woodgate R, Rajagopolan M, Lu C, Echols H (1989) UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD′. Proc Natl Acad Sci USA 86:7301–7305
Author information
Authors and Affiliations
Additional information
Communicated by M. Sekiguchi
Rights and permissions
About this article
Cite this article
Nowicka, A., Kanabus, M., Sledziewska-Gójska, E. et al. Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli . Molec. Gen. Genet. 243, 584–592 (1994). https://doi.org/10.1007/BF00284207
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00284207