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. 2005 Mar;25(6):2297-309.
doi: 10.1128/MCB.25.6.2297-2309.2005.

DNA interstrand cross-link repair in the Saccharomyces cerevisiae cell cycle: overlapping roles for PSO2 (SNM1) with MutS factors and EXO1 during S phase

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DNA interstrand cross-link repair in the Saccharomyces cerevisiae cell cycle: overlapping roles for PSO2 (SNM1) with MutS factors and EXO1 during S phase

Louise J Barber et al. Mol Cell Biol. 2005 Mar.

Abstract

Pso2/Snm1 is a member of the beta-CASP metallo-beta-lactamase family of proteins that include the V(D)J recombination factor Artemis. Saccharomyces cerevisiae pso2 mutants are specifically sensitive to agents that induce DNA interstrand cross-links (ICLs). Here we establish a novel overlapping function for PSO2 with MutS mismatch repair factors and the 5'-3' exonuclease Exo1 in the repair of DNA ICLs, which is confined to S phase. Our data demonstrate a requirement for NER and Pso2, or Exo1 and MutS factors, in the processing of ICLs, and this is required prior to the repair of ICL-induced DNA double-strand breaks (DSBs) that form during replication. Using a chromosomally integrated inverted-repeat substrate, we also show that loss of both pso2 and exo1/msh2 reduces spontaneous homologous recombination rates. Therefore, PSO2, EXO1, and MSH2 also appear to have overlapping roles in the processing of some forms of endogenous DNA damage that occur at an irreversibly collapsed replication fork. Significantly, our analysis of ICL repair in cells synchronized for each cell cycle phase has revealed that homologous recombination does not play a major role in the direct repair of ICLs, even in G2, when a suitable template is readily available. Rather, we propose that recombination is primarily involved in the repair of DSBs that arise from the collapse of replication forks at ICLs. These findings have led to considerable clarification of the complex genetic relationship between various ICL repair pathways.

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Figures

FIG. 1.
FIG. 1.
Analyses of the sensitivities of combinations of pso2, rad1, rad4, rad27, mre11, exo1, and msh2 gene disruptions (derived from B356-7C) treated with HN2. All results are means from at least three independent experiments. Error bars, standard errors of the means.
FIG. 2.
FIG. 2.
Repair of DSBs in HN2-treated pso2, pso2 exo1, pso2 msh2, pso2 exo1 msh2, and rad4 cells. Exponentially growing cells were either treated with 50 μM HN2 for 1 h at 28°C or mock treated (U) with water and were subsequently allowed to repair in minimal medium for 2, 4, or 24 h. The U sample was allowed to repair for 24 h. Samples were analyzed on contour-clamped homogeneous electric field gels.
FIG. 3.
FIG. 3.
Relative sensitivities of disruptant strains (derived from B356-7C) to 100 μM HN2 added in G1, S, or G2 phase. All results are means from at least three independent experiments. Error bars, standard errors of the means.
FIG. 4.
FIG. 4.
(A) Map of the inverted-repeat substrate. (B) Mean recombination rates between the ade2 inverted repeats, determined from at least nine independent colonies per strain. Error bars, standard errors of the means.
FIG. 5.
FIG. 5.
HN2 sensitivities of pso2, msh3, msh6, and mlh1 strains and cognate multiple disruptants (derived from B356-7C) treated with HN2.
FIG. 6.
FIG. 6.
Roles of NER, Pso2, MutS-Exo1 factors, and homologous recombination in processing of intermediates generated at ICLs in different phases of the yeast cell cycle. TLS, translesion synthesis; H.R., homologous recombination.

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