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.…”

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.…”

Dissecting the cell cycle regulation, DNA damage sensitivity and lifespan effects of caffeine in fission yeast

“…Furthermore, Rhp6 has been shown to mediate the cell cycle effects of caffeine (Rowley and Zhang, 1999). Caffeine may thus interfere with Rhp6- and Cut8-mediated DNA damage repair (Kearsey et al, 2007; Rowley and Zhang, 1999; Takeda and Yanagida, 2005). We have confirmed that caffeine even at 10mM did not drive rhp6 Δ mutants, previously exposed to HU, into mitosis (Figure 7A).…”

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 ).…”

“…We have used DNA combing in fission yeast to systematically look at the effect of the checkpoint on individual origins and forks at a global scale and hence to capture the heterogeneity in response to damage. To understand the behavior of the checkpoint at a global and local scale we have used three different types of lesion inducing agents at vastly differing concentrations—3.5mM (0.03%) for MMS, 1μM for 4NQO and 16.5 μM for bleomycin—which thus produce very different densities of lesions—1000 per Mb for MMS, 40 per Mb for 4NQO and 20 per Mb for bleomycin ( S1 Fig ) [ 53 – 56 ]—but nonetheless have very similar effects on bulk replication kinetics ( Fig 3 , S3 and S4 Figs). We find that the inhibition of origin firing is a checkpoint-dependent response, but one that develops more slowly in response to the many fork encounters with MMS than the less frequent but more severe fork interactions with 4NQO lesions or in response to repair of bleomycin-induced double-strand breaks.…”

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.…”

Dissecting the cell cycle regulation, DNA damage sensitivity and lifespan effects of caffeine in fission yeast

“…Furthermore, Rhp6 has been shown to mediate the cell cycle effects of caffeine (Rowley and Zhang, 1999). Caffeine may thus interfere with Rhp6- and Cut8-mediated DNA damage repair (Kearsey et al, 2007; Rowley and Zhang, 1999; Takeda and Yanagida, 2005). We have confirmed that caffeine even at 10mM did not drive rhp6 Δ mutants, previously exposed to HU, into mitosis (Figure 7A).…”

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 ).…”

“…We have used DNA combing in fission yeast to systematically look at the effect of the checkpoint on individual origins and forks at a global scale and hence to capture the heterogeneity in response to damage. To understand the behavior of the checkpoint at a global and local scale we have used three different types of lesion inducing agents at vastly differing concentrations—3.5mM (0.03%) for MMS, 1μM for 4NQO and 16.5 μM for bleomycin—which thus produce very different densities of lesions—1000 per Mb for MMS, 40 per Mb for 4NQO and 20 per Mb for bleomycin ( S1 Fig ) [ 53 – 56 ]—but nonetheless have very similar effects on bulk replication kinetics ( Fig 3 , S3 and S4 Figs). We find that the inhibition of origin firing is a checkpoint-dependent response, but one that develops more slowly in response to the many fork encounters with MMS than the less frequent but more severe fork interactions with 4NQO lesions or in response to repair of bleomycin-induced double-strand breaks.…”

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.…”

Dissecting the cell cycle regulation, DNA damage sensitivity and lifespan effects of caffeine in fission yeast

“…Furthermore, Rhp6 has been shown to mediate the cell cycle effects of caffeine (Rowley and Zhang, 1999). Caffeine may thus interfere with Rhp6- and Cut8-mediated DNA damage repair (Kearsey et al, 2007; Rowley and Zhang, 1999; Takeda and Yanagida, 2005). We have confirmed that caffeine even at 10mM did not drive rhp6 Δ mutants, previously exposed to HU, into mitosis (Figure 7A).…”

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 ).…”

“…We have used DNA combing in fission yeast to systematically look at the effect of the checkpoint on individual origins and forks at a global scale and hence to capture the heterogeneity in response to damage. To understand the behavior of the checkpoint at a global and local scale we have used three different types of lesion inducing agents at vastly differing concentrations—3.5mM (0.03%) for MMS, 1μM for 4NQO and 16.5 μM for bleomycin—which thus produce very different densities of lesions—1000 per Mb for MMS, 40 per Mb for 4NQO and 20 per Mb for bleomycin ( S1 Fig ) [ 53 – 56 ]—but nonetheless have very similar effects on bulk replication kinetics ( Fig 3 , S3 and S4 Figs). We find that the inhibition of origin firing is a checkpoint-dependent response, but one that develops more slowly in response to the many fork encounters with MMS than the less frequent but more severe fork interactions with 4NQO lesions or in response to repair of bleomycin-induced double-strand breaks.…”