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Preferential D-loop extension by a translesion DNA polymerase underlies error-prone recombination.

ABSTRACT: Although homologous recombination is considered an accurate form of DNA repair, genetics suggest that the Escherichia coli translesion DNA polymerase IV (Pol IV, also known as DinB) promotes error-prone recombination during stress, which allows cells to overcome adverse conditions. However, how Pol IV functions and is regulated during recombination under stress is unknown. We show that Pol IV is highly proficient in error-prone recombination and is preferentially recruited to displacement loops (D loops) at stress-induced concentrations in vitro. We also found that high-fidelity Pol II switches to exonuclease mode at D loops, which is stimulated by topological stress and reduced deoxyribonucleotide pool concentration during stationary phase. The exonuclease activity of Pol II enables it to compete with Pol IV, which probably suppresses error-prone recombination. These findings indicate that preferential D-loop extension by Pol IV facilitates error-prone recombination and explain how Pol II reduces such errors in vivo.

SUBMITTER: Pomerantz RT

PROVIDER: S-EPMC3685420 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Preferential D-loop extension by a translesion DNA polymerase underlies error-prone recombination.

Pomerantz Richard T RT Kurth Isabel I Goodman Myron F MF O'Donnell Mike E ME

Nature structural & molecular biology 20130519 6

Although homologous recombination is considered an accurate form of DNA repair, genetics suggest that the Escherichia coli translesion DNA polymerase IV (Pol IV, also known as DinB) promotes error-prone recombination during stress, which allows cells to overcome adverse conditions. However, how Pol IV functions and is regulated during recombination under stress is unknown. We show that Pol IV is highly proficient in error-prone recombination and is preferentially recruited to displacement loops ...[more]

PMID: 23686288

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Project description:DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The Δ1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1-25 variant, was ∼7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens.

Project description:UnlabelledEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication.ImportanceDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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Preferential D-loop extension by a translesion DNA polymerase underlies error-prone recombination.

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Preferential D-loop extension by a translesion DNA polymerase underlies error-prone recombination.

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