title: Pol III proofreading activity prevents lesion bypass as evidenced by its molecular signature within E.coli cells.
abstract: 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.
authors.affiliation: UPR 9003, CNRS Cancérogenèse et Mutagenèse Moléculaire et Structurale, Blvd S. Brant, 67400 Strasbourg, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Pages
authors.forename: Régine
authors.initials: R
authors.lastname: Janel-Bintz
authors.forename: Robert P
authors.initials: RP
authors.lastname: Fuchs
mesh_terms: 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
chemical_list: D004269:DNA, Bacterial; D004258:DNA Polymerase III