Advertisement

Molecular and General Genetics MGG

, Volume 251, Issue 4, pp 493–498 | Cite as

Analysis of the mutagenic properties of the UmuDC, MucAB and RumAB proteins, using a site-specific abasic lesion

  • C. W. Lawrence
  • A. Borden
  • R. Woodgate
Original Paper

Abstract

ThemucAB andrumAB loci have been shown to promote mutagenesis to a greater extent than the structurally and functionally homologousEscherichia coli umuDC operon. We have analyzed the basis of this enhanced mutagenesis by comparing the influence of these operons, relative toumuDC, on the mutagenic properties of each of two abasic sites, specifically located in a single-stranded vector. Experiments with these vectors are useful analytical tools because they provide independent estimates of the efficiency of translesion synthesis and of the relative frequencies of each type of nucleotide insertion or other kind of mutagenic event. TheumuDC, mucAB, andrumAB genes were expressed from their naturalLexA-regulated promoter on low-copy-number plasmids in isogenic strains carrying aumuDC deletion. In addition, plasmids expressing the UmuD'C, MucA'B, or RumA'B proteins were also used. Compared toumuDC, the chief effect ofmucAB was to increase the efficiency of translesion synthesis past the abasic site. The enhanced capacity ofmucAB for translesion synthesis depended about equally on an inherently greater capacity to promote this process and on a greater susceptibility of the MucA protein to proteolytic processing. The RumA protein also appeared to be more susceptible to proteolytic processing, but the inherent capacity of theRum products for translesion synthesis was no greater than that ofUmuDC. dAMP was inserted opposite one of the two abasic sites studied at a somewhat greater frequency in strains expressingrum (82%) compared to those expressingumu (72%), which might result in higher mutation frequencies inrumAB than inumuDC strains.

Key words

Escherichia coli umuDC mucAB rumAB Specifically located abasic site 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Banerjee SK, Borden A, Christensen RB, LeClerc JE, Lawrence CW (1990) SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells. J Bacteriol 172:2105–2112Google Scholar
  2. Blanco M, Herrera G, Collado P, Rebollo JE, Botella LM (1982) Influence of RecA protein on induced mutagenesis. Biochimie 64:633–636Google Scholar
  3. Blanco M., Herrera G, Aleixandre V (1986) Different efficiency of UmuDC and MucAB proteins in UV light-induced mutagenesis inEscherichia coli. Mol Gen Genet 205:234–239Google Scholar
  4. Churchward G, Belin D, Nagamine Y (1984) A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene 31:165–171Google Scholar
  5. Doyle N, Strike P (1995) The spectra of base substitutions induced by theimpCAB, mucAB andumuDC error-prone DNA repair operons differ following exposure to methyl methane sulfonate. Mol Gen Genet 247:735–741Google Scholar
  6. Ennis DG, Levine AS, Koch WH, Woodgate R (1995) Analysis ofrecA mutants with altered SOS functions. Mutat Res 336:39–48Google Scholar
  7. 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 90:8169–8173Google Scholar
  8. Foster PL, Sullivan AD (1988) Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response ofEscherichia coli. Mol Gen Genet 214:467–473Google Scholar
  9. Fowler RG, McGinty L, Mortelmans KE (1981) Mutational specificity of ultraviolet light inEscherichia coli with and without the plasmid pKM101. Genetics 99:25–40Google Scholar
  10. Gibbs PEM, Lawrence CW (1995) Novel mutagenic properties of an abasic site inSaccharomyces cerevisiae. J Mol Biol 251:229–236Google Scholar
  11. Hauser J, Levine AS, Ennis DG, Chumakov KM, Woodgate R (1992) The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein. J Bacteriol 174:6844–6851Google Scholar
  12. Ho C, Kulaeva OI, Levine AS, Woodgate R (1993) A rapid method for cloning mutagenic DNA repair genes: isolation ofumu-complementing genes from multidrug resistance plasmids R391, R446b, and R471a. J Bacteriol 175:5411–5419Google Scholar
  13. Kulaeva OI, Wooton JC, Levine AS, Woodgate R (1995) Characterization of theumu-complementing operon from R391. J Bacteriol 177:2737–2743Google Scholar
  14. Lawrence CW, Borden A, Banerjee SK, LeClerc JE (1990) Mutation frequency and spectrum resulting from a single abasic site in a single-stranded vector. Nucleic Acids Res 18:2153–2157Google Scholar
  15. Loeb LA, Preston BD (1986) Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet 20:201–230Google Scholar
  16. Mattern IE, Olthoff FP, Jacobs-Meijsing BLM, Enger-Valk BE, Pouwels PH, Lohman PHM (1985) A system to determine base pair substitutions at the molecular level based on restriction enzyme analysis; influence of themuc genes of pKM101 on the specificity of mutation induction inE. coli. Mutat Res 148:35–45Google Scholar
  17. McCann J, Spingarn NE, Kobori J, Ames BN (1975) Detection of carcinogenes as mutagens: bacterial tester strains with R factor plasmids. Proc Natl Acad Sci USA 72:979–983Google Scholar
  18. Palejwala VA, Rzepka RW, Humayun MZ (1993) UV irradiation ofEscherichia coli modulates mutagenesis at a site-specific ethenocytosine residue on M13 DNA. Biochemistry 32:4112–4120Google Scholar
  19. Perry KL, Elledge SJ, Mitchell BB, Marsh L, Walker GC (1985)umuDC andmucAB operons whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology. Proc Natl Acad Sci USA 82:4331–4335Google Scholar
  20. Strauss BS (1991) The ‘A rule’ of mutagen specificity: a consequence of DNA polymerase bypass of non-instructional lesions? Bio-Essays 13:79–84Google Scholar
  21. Szekeres ES, Woodgate R, Lawrence CW (1995) Substitution ofmucAB orrumAB forumuDC alters the relative frequency of the two classes of mutations induced by a site-specific T-T cyclobutane dimer and also the efficiency of translesion synthesis. J Bacteriol 178:2559–2563Google Scholar
  22. Tadmor Y, Ascarelli-Goeli R, Skaliter R, Livneh Z (1992) Over-production of the β subunit of DNA polymerase III holoenzyme reduces UV mutagenesis inEscherichia coli. J Bacteriol 174:2517–2524Google Scholar
  23. Urios A, Herrera G, Aleixandre V, Sommer S, Blanco M (1994) Mutability ofSalmonella tester strains TA1538 (hisD3502) and TA1535 (hisG46) containing the UmuD' and UmuC proteins ofEscherichia coli. Environ Mol Mutagen 23:63–67Google Scholar
  24. Watanabe M, Nohmi T, Ohta T (1994) Effects of theumuDC, mucAB, andsamAB operons on the mutagenic specificity of chemical mutagenesis inEscherichia coli. II. Base substitution mutagenesis. Mutat Res 314:39–49Google Scholar
  25. Woodgate R (1992) Construction of aumuDC operon substitution mutation inEscherichia coli. Mutat Res 281:221–225Google Scholar
  26. Woodgate R, M Rajagopalan, C Lu and H Echols (1989) UmuC mutagenesis protein ofEscherichia coli: purification and interaction with UmuD and UmuD'. Proc Natl Acad Sci USA 86: 7301–7305Google Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • C. W. Lawrence
    • 1
  • A. Borden
    • 1
  • R. Woodgate
    • 2
  1. 1.Department of BiophysicsUniversity of Rochester Medical CenterRochesterUSA
  2. 2.Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human DevelopmentNational Institutes of HealthBethesdaUSA

Personalised recommendations