No 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

Cells have evolved checkpoint responses to arrest or delay the cell cycle, activate DNA repair networks, or induce apoptosis after genomic perturbation. Cells have also evolved the translesion synthesis processes to tolerate genomic lesions by either error-free or error-prone repair. Here, we show that after a replication perturbation, cells exhibit a mutator phenotype, which can be significantly affected by mutations in the checkpoint elements Cds1 and Rad17 or translesion synthesis polymerases DinB and Pol. Cells respond to genomic perturbation by up-regulation of DinB in a checkpoint activation-dependent manner. Moreover, association of DinB with chromatin is dependent on functional Rad17, and DinB physically interacts with the checkpoint-clamp components Hus1 and Rad1. Thus, translesion synthesis is a part of the checkpoint response.

No abstract

DNA polymerase ␦ (pol ␦) plays an essential role in DNA replication, repair, and recombination. We have purified pol ␦ from Schizosaccharomyces pombe more than 10 3 -fold and demonstrated that the polymerase activity of purified S. pombe pol ␦ is completely dependent on proliferating cell nuclear antigen and replication factor C. SDS͞ PAGE analysis of the purified fraction indicated that the pol ␦ complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa. Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively. The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides. The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive cdc1-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on S. pombe chromosome 1.