Fission Yeast Cut8 Is Required for the Repair of DNA Double-Strand Breaks, Ribosomal DNA Maintenance, and Cell Survival in the Absence of Rqh1 Helicase

Abstract

Schizosaccharomyces pombe Rqh1 is a member of the RecQ DNA helicase family. Members of this protein family are mutated in cancer predisposition diseases, causing Bloom's, Werner, and Rothmund-Thomson syndromes. Rqh1 forms a complex with topoisomerase III and is proposed to process or disrupt aberrant recombination structures that arise during S phase to allow proper chromosome segregation during mitosis. Intriguingly, in the absence of Rqh1, processing of these structures appears to be dependent on Rad3 (human ATR) in a manner that is distinct from its role in checkpoint control. Here, we show that rad3 rqh1 mutants are normally committed to a lethal pathway of DNA repair requiring homologous recombination, but blocking this pathway by Rhp51 inactivation restores viability. Remarkably, viability is also restored by overexpression of Cut8, a nuclear envelope protein involved in tethering and proper function of the proteasome. In keeping with a recently described function of the proteasome in the repair of DNA double-strand breaks, we found that Cut8 is also required for DNA double-strand break repair and is essential for proper chromosome segregation in the absence of Rqh1, suggesting that these proteins might function in a common pathway in homologous recombination repair to ensure accurate nuclear division in S. pombe.Eukaryotic cells engage a variety of protective responses following DNA damage. These include enzymes which repair specific DNA lesions after DNA damage as well as regulators that coordinate DNA repair and cell cycle progression. Mutations abolishing many of these processes result in genome instability and cause cancer-prone syndromes in humans. One important class of these syndromes is caused by defects in RecQ helicases (1). These enzymes unwind DNA in a 3Ј to 5Ј direction and are proposed to function in DNA recombination repair. Another example of this is the phosphatidylinositol 3-kinase-like protein kinases ATM (mutated in ataxia telangiectasia) (12) and ATR (ATM and Rad3 related). These proteins are associated with DNA damage surveillance and control of cell cycle checkpoints. Three checkpoint protein complexes have been suggested to play roles in sensing DNA damage. The components of these complexes in Schizosaccharomyces pombe are the checkpoint Rad proteins, including the Rad3-Rad26 complex (human ATR and ATRIP, respectively), the PCNA-like Rad9-Rad1-Hus1 (9-1-1) complex, and the Rad17-replication factor C clamp loader complex (4). They are involved in the activation of two downstream effector kinases, Chk1 and Cds1 (19,29). The Cds1-dependent S-phase checkpoint is required for arresting the cell cycle, stabilizing replication forks, and preventing late origin firing. The Chk1-dependent DNA damage checkpoint prevents entry into mitosis until the completion of DNA repair.DNA double-strand breaks (DSBs) are the most severe damage to DNA caused by environmental factors and occur spontaneously during normal cellular metabolism. In eukaryotes, DSBs are repaired by homologous recombination (HR)...

Redundant roles of Srs2 helicase and replication checkpoint in survival and rDNA maintenance in Schizosaccharomyces pombe

“…4b, circles with solid lines) out of 44 tetrads for each diploid clone. This was in marked contrast to most of the srs2-related synthetic lethality in S. cerevisiae (Aboussekhra et al 1992;Schild 1995;GangloV et al 2000) or the rqh1 rad3 in S. pombe (Kearsey et al 2007). We also conWrmed the severe growth defect of srs2 rhp51 and srs2 rad22 (Fig.…”

“…5c-e, Figure S2). Kearsey et al (2007) reported a strange behavior of chromosome III of the rad3 cells in PFGE probably due to segregation failure. This was conWrmed in the rad3 strains (data not shown), but chromosome III of the rad3 ts cells did not show such an abnormality on temperature shift (Fig.…”

Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA

“…G1 synchronized cells were released into S phase with or without DNA damage, using the commonly used dose of 3.5 mM (0.03%) MMS or a dose of 1 μM 4NQO or 16.5 μM bleomycin chosen to produce a similar slowing of bulk replication ( S3 and S4 Figs). These doses of MMS, 4NQO and bleomycin cause lesions about once every 1 kb, 25 kb, and 50 kb respectively ( S1 Fig ) [ 53 – 56 ]. By flow cytometry, control cells completed replication by 80 minutes, while in the presence of either drug cells slowed replication, reaching only about 60% replicated by the end of the time course ( Fig 3A and S5A Fig ).…”

“…MMS and 4NQO create polymerase-stalling lesions and have been shown to activate the intra-S checkpoint [ 17 , 44 , 49 – 52 ]. The standard dose of 3.5 mM (0.03%) MMS causes about one lesion every 1 kb, whereas a physiologically similar dose of 1 μM 4NQO in CHO cells causes one lesion about every 25 kb and we estimate 16.5 μM of bleomycin causes about one double-strand break per 50 kb ( S1 Fig ) [ 53 – 56 ]. Although these are only rough approximations of lesion density, they show that forks will encounter many more MMS lesions than 4NQO lesions or bleomycin-induced double-strand breaks.…”

Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8

“…Additional data showing that Cut8 is essential for nuclear localization is the recent finding that a reduction in Cut8 levels coincides with the altered localization of the proteasome from the nucleus to the cytoplasm upon the transition from vegetative proliferation to the G0/quiescent phase (22). Nuclear sequestration of the proteasome by Cut8 has also been shown to be critical for double-strand break repair (23). This is likely due to the requirement of the proteasome for cohesion cleavage.…”