Pol III proofreading activity prevents lesion bypass as evidenced by its molecular signature within E.coli cells.
Replication of genomes that contain blocking DNA lesions entails the transient replacement of the replicative DNA polymerase (Pol) by a polymerase specialized in lesion bypass. Here, we isolate and visualize at nucleotide resolution level, replication intermediates formed during lesion bypass of a single N-2-acetylaminofluorene-guanine adduct (G-AAF) in vivo. In a wild-type strain, a ladder of replication intermediates mapping from one to four nucleotides upstream of the lesion site, can be observed. In proofreading-deficient strains (mutD5 or dnaQ49), these replication intermediates disappear, thus assigning the degradation ladder to the polymerase-associated exonuclease activity. Moreover, in mutD5, a new band corresponding to the insertion of a nucleotide opposite to the lesion site is observed, suggesting that the polymerase and exonuclease activities of native Pol III enter a futile insertion-excision cycle that prevents translesion synthesis. The bypass of the G-AAF adduct located within the NarI sequence context requires the induction of the SOS response and involves either Pol V or Pol II in an error-free or a frameshift pathway, respectively. In the frameshift mutation pathway, inactivation of the proofreading activity obviates the need for SOS induction but nonetheless necessitates a functional polB gene, suggesting that, although proofreading-deficient Pol III incorporates a nucleotide opposite G-AAF, further extension still requires Pol II. These data are corroborated using a colony-based bypass assay.
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Vincent
V
UPR 9003, CNRS Cancérogenèse et Mutagenèse Moléculaire et Structurale, Blvd S. Brant, 67400 Strasbourg, France.
Janel-Bintz
Régine
R
Fuchs
Robert P
RP
D000483:Alleles; D001483:Base Sequence; D004249:DNA Damage; D004258:DNA Polymerase III / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016368:Frameshift Mutation; D005798:Genes, Bacterial
D004269:DNA, Bacterial; D004258:DNA Polymerase III