Abundance of Prereplicative Complexes (Pre-RCs) Facilitates Recombinational Repair under Replication Stress in Fission Yeast

Kentaro Maki,Hiroko Hashizume,Takahiro Inoue,Yuko Kobayashi,Chikako Shigaki,Atsushi Onaka,Hisao Masukata,Naoko Somete,Shigefumi Murakami,Tatsuro S Takahashi,Takuro Nakagawa

doi:10.1074/jbc.m111.285619

Abstract

Mcm2-7 complexes are loaded onto chromatin with the aid of Cdt1 and Cdc18/Cdc6 and form prereplicative complexes (pre-RCs) at multiple sites on each chromosome. Pre-RCs are essential for DNA replication and surviving replication stress. However, the mechanism by which pre-RCs contribute to surviving replication stress is largely unknown. Here, we isolated the fission yeast mcm6-S1 mutant that was hypersensitive to methyl methanesulfonate (MMS) and camptothecin (CPT), both of which cause forks to collapse. The mcm6-S1 mutation impaired the interaction with Cdt1 and decreased the binding of minichromosome maintenance (MCM) proteins to replication origins. Overexpression of Cdt1 restored MCM binding and suppressed the sensitivity to MMS and CPT, suggesting that the Cdt1-Mcm6 interaction is important for the assembly of pre-RCs and the repair of collapsed forks. MMS-induced Chk1 phosphorylation and Rad22/Rad52 focus formation occurred normally, whereas cells containing Rhp54/Rad54 foci, which are involved in DNA strand exchange and dissociation of the joint molecules, were increased. Remarkably, G(1) phase extension through deletion of an S phase cyclin, Cig2, as well as Cdt1 overexpression restored pre-RC assembly and suppressed Rhp54 accumulation. A cdc18 mutation also caused hypersensitivity to MMS and CPT and accumulation of Rhp54 foci. These data suggest that an abundance of pre-RCs facilitates a late step in the recombinational repair of collapsed forks in the following S phase.

Highlights

Of many prereplicative complexes is important for DNA replication and surviving replication stress
As expected from previous studies [64], a deletion of the damage checkpoint kinase Chk1 resulted in hypersensitivity to methyl methanesulfonate (MMS) and CPT, whereas the deletion of the replication checkpoint kinase Cds1 did not affect the sensitivity to these agents compared with wild type, demonstrating that treatment with MMS and CPT initiates the damage checkpoint pathway
In contrast to the mcm4-c84 mutant, the mcm6-S1 mutant was hypersensitive to MMS and CPT, suggesting that the repair of collapsed forks is impaired in the mcm6-S1 mutant. mcm6-S1 cells were only slightly sensitive to chronic exposure to HU

Summary

Background

Of many prereplicative complexes (pre-RCs) is important for DNA replication and surviving replication stress. Overexpression of Cdt restored MCM binding and suppressed the sensitivity to MMS and CPT, suggesting that the Cdt1-Mcm interaction is important for the assembly of pre-RCs and the repair of collapsed forks. A cdc mutation caused hypersensitivity to MMS and CPT and accumulation of Rhp foci These data suggest that an abundance of pre-RCs facilitates a late step in the recombinational repair of collapsed forks in the following S phase. A mutation in the MCM loader Cdc caused hypersensitivity to MMS and CPT and Rhp accumulation These data suggest that the assembly of many pre-RCs facilitates the late step in the recombinational repair of collapsed forks. We propose a model in which the forks converging at one-ended DSBs facilitate the late step in SDSA by providing a second DSB end

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION