Pol III proofreading activity prevents lesion bypass as evidenced by its molecular signature within E.coli cells.
J Mol Biol
; 352(3): 501-9, 2005 Sep 23.
Article
en En
| MEDLINE
| ID: mdl-16111701
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.
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Banco de datos:
MEDLINE
Asunto principal:
ADN Polimerasa III
/
Reparación del ADN
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Escherichia coli
Idioma:
En
Año:
2005
Tipo del documento:
Article