Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III.
The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli generates the induced, multigene SOS response. One component of the SOS response is a marked increase in mutation rate, dependent on RecA protein and the induced mutagenesis proteins UmuC and UmuD. A variety of previous indirect approaches have indicated that SOS mutagenesis results from replicative bypass of the DNA lesion by DNA polymerase III (pol III) holoenzyme in a reaction mediated by RecA, UmuC, and a processed form of UmuD termed UmuD'. To study the biochemistry of SOS mutagenesis, we have reconstituted replicative bypass with a defined in vitro system containing purified protein and a DNA substrate with a single abasic DNA lesion. The replicative bypass reaction requires pol III, UmuC, UmuD', and RecA. The nonprocessed UmuD protein does not replace UmuD' but inhibits the bypass activity of UmuD', perhaps by sequestering UmuD' in a heterodimer. Our experiments demonstrate directly that the UmuC-UmuD' complex and RecA act to rescue an otherwise stalled pol III holoenzyme at a replication-blocking DNA lesion.
Rajagopalan
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Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
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Woodgate
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O'Donnell
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Goodman
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Echols
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D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004249:DNA Damage; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008961:Models, Structural; D011938:Rec A Recombinases / Q000302:isolation & purification / Q000378:metabolism*; D013014:SOS Response, Genetics; D013698:Templates, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; D011938:Rec A Recombinases; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli