Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress

Abstract

. Upon S-phase arrest by acute hydroxyurea treatment, Mus81 is not required for cell viability but is essential for recovery from replication fork collapse. Moreover, Mus81 undergoes extensive Cds1-dependent phosphorylation and dissociates from chromatin in hydroxyurea-arrested cells, thereby preventing it from cleaving stalled replication forks that could lead to fork breakage and chromosomal rearrangement. These results provide novel insights into how Cds1 regulates Mus81 accordingly when cells experience different replication stress to preserve genome integrity.[Keywords: Genome stability; DNA replication; replication checkpoint; Cds1; Mus81-Eme1; recombination] DNA replication is a process fraught with danger for the integrity of genome Kolodner et al. 2002;Osborn et al. 2002;Muzi-Falconi et al. 2003). DNA lesions, deoxyribonucleotide (dNTP) depletion, and defective replication proteins are among the many events that can impede replication fork progression. Stalled forks are thought to pose serious threats to genomic integrity because they are barriers to completion of DNA replication and they are unstable structures that can collapse, rearrange, or break (McGlynn and Lloyd 2002). These events can lead to chromosomal rearrangements and deleterious genomic deletions. These genome-destabilizing occurrences are associated with many human diseases, including cancer.Preservation of genome integrity during replication fork arrest is the responsibility of the replication checkpoint (Elledge 1996;Lopes et al. 2001;Tercero and Diffley 2001;Carr 2002;Osborn et al. 2002;Sogo et al. 2002;Cobb et al. 2003). This genome maintenance system delays the onset of mitosis, thereby providing time to complete replication before cell division. The overall understanding of replication checkpoints is most advanced in studies of budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe (Lindsay et al. 1998;Lopes et al. 2001;Tercero and Diffley 2001;Osborn et al. 2002;Sogo et al. 2002;Kai and Wang 2003a;Longhese et al. 2003). An important function of the replication checkpoint is to control the stability of stalled forks (Murakami and Okayama 1995;Boddy et al. 1998Boddy et al. , 2003Lindsay et al. 1998;Lopes et al. 2001;Tercero and Diffley 2001;Sogo et al. 2002) and the association of the replisome with the replication fork (Cobb et al. 2003;Lucca et al. 2004;Cotta-Ramusino et al. 2005). A protein kinase called Rad53 in budding yeast and Cds1 in fission yeast is the critical transducer of the replication checkpoint. Mutants defective for the kinase are acutely sensitive to hydroxyurea (HU), a chemical that depletes cellular dNTP pools by inhibiting ribonucleotide reductase (Reichard 1988;Lopes et al. 2001). HU treatment of these mutants leads to formation of pathological DNA structures such as regressed forks and hemi-replicated DNA regions, as well as broken forks Cold Spring Harbor Laboratory Press on May 9, 2018 -Published by genesdev.cshlp.org Downloaded from

The Mek1 phosphorylation cascade plays a role in meiotic recombination ofSchizosaccharomyces pombe

“…Mek1 appears to be primarily responsible for the T218 and T422 phosphorylations of Mus81 as judged by the greater efficiency of 32 P incorporation by Mek1 than by Cds1, at least in vitro, when compared with the relative intensities of the Simply Bluestained bands (white arrows and arrowheads in Sup. Fig.…”

“…It was previously shown that Mus81 is phosphorylated by an unknown kinase on T239 (now T275) in the T-X-X-D motif in response to HU-induced replication arrest. 25,32 Here, we referred to their T239 as T275, according to the revised DNA sequence in the S. pombe database (http://www.genedb.org/genedb/Sear ch?name=SPCC4G3.05c&organism=pombe). We propose here that this unknown kinase is most probably Cds1 and/or Mek1, because either could directly phosphorylate Mus81 on T275 in vitro (Sup .…”

“…This interaction, which removes Mus81 from chromatin, thus negatively regulates the Mus81 function in DNA cleavage. 32 Moreover, the recombination frequency in mus81.T239A cells after acute exposure to HU was more enhanced than in WT cells, 32 which could also happen during meiosis. Since Mus81-T275 negatively regulates DNA cleavage by removing Mus81 from chromatin, and the T275A mutation caused abnormal recombination, 32 our results (Sup.…”

“…1B and lane 8). Moreover, addition of wortmannin, an inhibitor of ATM/ATR and Rad3 kinase activity, reduced the amount of 32 P incorporation in a dose-dependent manner (Sup. Fig.…”

“…Subsequently, activated Mek1 phosphorylates Mus81 on T275, which negatively regulates the Mus81 function. 32 Furthermore, Mek1 phosphorylates Rdh54 on T6 and T673, which appears to have a regulatory role in meiotic progression.…”