Repair of chromosomal abasic sites in vivo involves at least three different repair pathways.
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.
Otterlei
M
M
Institute of Cancer Research and Molecular Biology, The Faculty of Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
Kavli
B
B
Standal
R
R
Skjelbred
C
C
Bharati
S
S
Krokan
H E
HE
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003062:Codon / Q000235:genetics; D045647:DNA Glycosylases; D004265:DNA Helicases; D004252:DNA Mutational Analysis; D004260:DNA Repair / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D009154:Mutation / Q000235:genetics; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011995:Recombination, Genetic / Q000235:genetics; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase
D001426:Bacterial Proteins; D003062:Codon; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D004259:DNA-Directed DNA Polymerase; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase