Escherichia coli responses to a single DNA adduct.
To study the mechanisms by which Escherichia coli modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1, N(6)-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF, recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.
Pandya
G A
GA
Laboratory of Chemical Biology, Department of Pharmacological Sciences, SUNY at Stony Brook, Stony Brook, New York 11794-8651, USA.
Yang
I Y
IY
Grollman
A P
AP
Moriya
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018736:DNA Adducts / Q000187:drug effects*; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003839:Deoxyadenosines / Q000494:pharmacology; D004706:Endodeoxyribonucleases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial / Q000502:physiology; D008969:Molecular Sequence Data; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013014:SOS Response, Genetics
D001426:Bacterial Proteins; D018736:DNA Adducts; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; C081006:RecO protein, E coli; C489318:RecR protein, E coli; C061260:RuvB protein, Bacteria; C489340:recF protein, E coli; C069806:ruvC protein, E coli; C065831:RecR protein, Bacteria; C071446:RecG protein, E coli; C032221:1,N(6)-ethenodeoxyadenosine; C066218:recF protein, Bacteria; D011938:Rec A Recombinases; D004258:DNA Polymerase III; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases