Repair of chromosomal abasic sites in vivo involves at least three different repair pathways - PubMed (original) (raw)

Repair of chromosomal abasic sites in vivo involves at least three different repair pathways

M Otterlei et al. EMBO J. 2000.

Abstract

We introduced multiple abasic sites (AP sites) in the chromosome of repair-deficient mutants of Escherichia coli, in vivo, by expressing engineered variants of uracil-DNA glycosylase that remove either thymine or cytosine. After introduction of AP sites, deficiencies in base excision repair (BER) or recombination were associated with strongly enhanced cytotoxicity and elevated mutation frequencies, selected as base substitutions giving rifampicin resistance. In these strains, increased fractions of transversions and untargeted mutations were observed. In a recA mutant, deficient in both recombination and translesion DNA synthesis (TLS), multiple AP sites resulted in rapid cell death. Preferential incorporation of dAMP opposite a chromosomal AP site ('A rule') required UmuC. Furthermore, we observed an 'A rule-like' pattern of spontaneous mutations that was also UmuC dependent. The mutation patterns indicate that UmuC is involved in untargeted mutations as well. In a UmuC-deficient background, a preference for dGMP was observed. Spontaneous mutation spectra were generally strongly dependent upon the repair background. In conclusion, BER, recombination and TLS all contribute to the handling of chromosomal AP sites in E.coli in vivo.

PubMed Disclaimer

Figures

None

Fig. 1. Specificity of engineered DNA glycosylases. In vitro excision of adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) by CDG, TDG and UDG, from double-stranded DNA after incubation for 3 h.

None

Fig. 2. The cytotoxic effect of TDG expression in wild-type E.coli (filled squares) compared with expression in strains deficient in (A) UmuC (filled diamonds), RuvA (filled stars) and RecA (filled circles), or (B) endonuclease IV (open diamonds), exonuclease III (open stars) or endonuclease IV/exonuclease III (open circles). (C) Mutation frequency (rifR bacteria per 108 ampR bacteria) spontaneously (grey bars) and after induction of AP sites with CDG (black bars) or TDG (white bars). The n values indicate the number of experiments for each column and bars indicate standard deviations.

None

Fig. 3. Mutation spectra in the rpoB gene (RNA polymerase β) within codons 509–574 from rifR colonies, spontaneous (VEC) and after expression of UDG (only wild type), CDG or TDG. Codon positions are given on the X axis, and the number of mutations are given on the Y axis. The n value at the top left corner gives the total number of sequences. The mutations are classified as GC transitions (black bars), AT transitions (white bars), GC transversions (checked bars) and AT transversions (striped bars). The type of transitions and transversions, given as numbers as well as percentages, are specified at the top right corner of the spectra. In the recA mutant, only the spontaneous mutation spectrum was analysed.

Similar articles

Cited by

References

    1. Asai T. and Kogoma,T. (1994) Roles of ruvA, ruvC and recG gene functions in normal and DNA damage-inducible replication of the Escherichia coli chromosome. Genetics, 137, 895–902. - PMC - PubMed
    1. Berdal K.G., Johansen,R.F. and Seeberg,E. (1998) Release of normal bases from intact DNA by a native DNA repair enzyme. EMBO J., 17, 363–367. - PMC - PubMed
    1. Blatny J.M., Brautaset,T., Winther-Larsen,H.C., Karunakaran,P. and Valla,S. (1997) Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid, 38, 35–51. - PubMed
    1. Brotcorne-Lannoye A. and Maenhaut-Michel,G. (1986) Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene. Proc. Natl Acad. Sci. USA, 83, 3904–3908. - PMC - PubMed
    1. Christensen J.R., LeClerc,J.E., Tata,P.V., Christensen,R.B. and Lawrence,C.W. (1988) UmuC function is not essential for the production of all targeted lacI mutations induced by ultraviolet light. J. Mol. Biol., 203, 635–641. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources