PMID:16111701
| Citation |
Pages, V, Janel-Bintz, R and Fuchs, RP (2005) Pol III proofreading activity prevents lesion bypass as evidenced by its molecular signature within E.coli cells. J. Mol. Biol. 352:501-9 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1016/j.jmb.2005.07.063 |
| Keywords |
Alleles; Base Sequence; DNA Damage; DNA Polymerase III/metabolism; DNA Repair; DNA Replication; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Frameshift Mutation; Genes, Bacterial |
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