Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance

Bjergbæk, Lotte; Cobb, Jennifer A.; Tsai‐Pflugfelder, Monica; Gasser, Susan M.

doi:10.1038/sj.emboj.7600511

Cited by 136 publications

(151 citation statements)

References 51 publications

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“…Therefore, we speculate that the extent of checkpoint activation after MMS treatment varies in cells depending on whether Top3 is absent (as in top3 cells), catalytically active with normal turnover (as in wild-type cells), or catalytically dead and unable to turnover (as in cells overexpressing TOP3 Y356F ). Although this model is consistent with our data, we cannot rule out the possibility that the effects we observed could also be explained either by Top3 influencing events at stalled replication forks (e.g., the processing of stalled forks; Hishida et al, 2004), or the stabilization of DNA polymerases at stalled forks (Bjergbaek et al, 2005)], or by the fact that persistent X-molecules in top3 cells represent different DNA structures from those in cells overexpressing TOP3 Y356F .…”

Section: Discussionsupporting

confidence: 60%

“…It is worth noting that deletion of either TOP3 or RMI1 adversely affects the stability of Sgs1 and causes a reduction in Sgs1 protein levels (Chang et al, 2005). It is possible, therefore, that Sgs1 mediates the activation of Rad53 after DNA damage via its ability to physically interact with Rad53 (Frei and Gasser, 2000;Bjergbaek et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

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Top3 Processes Recombination Intermediates and Modulates Checkpoint Activity after DNA Damage

Mankouri

Hickson

2006

MBoC

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Mutation of TOP3 in Saccharomyces cerevisiae causes poor growth, hyperrecombination, and a failure to fully activate DNA damage checkpoints in S phase. Here, we report that overexpression of a dominant-negative allele of TOP3, TOP3 Y356F , which lacks the catalytic (decatenation) activity of Top3, causes impaired S-phase progression and the persistence of abnormal DNA structures (X-shaped DNA molecules) after exposure to methylmethanesulfonate. The impaired S-phase progression is due to a persistent checkpoint-mediated cell cycle delay and can be overridden by addition of caffeine. Hence, the catalytic activity of Top3 is not required for DNA damage checkpoint activation, but it is required for normal S-phase progression after DNA damage. We also present evidence that the checkpoint-mediated cell cycle delay and persistence of X-shaped DNA molecules resulting from overexpression of TOP3 Y356F are downstream of Rad51 function. We propose that Top3 functions in S phase to both process homologous recombination intermediates and modulate checkpoint activity. INTRODUCTIONTopoisomerases are highly conserved proteins that catalyze topological rearrangements in the structure of DNA and are required for many aspects of DNA metabolism. Of the three topoisomerases in yeast, the function(s) of the sole type IA topoisomerase, Top3, remains most poorly understood. However, it is likely that Top3 fulfills important roles in vivo, because deletion of TOP3 in Saccharomyces cerevisiae causes hyperrecombination, sensitivity to genotoxic agents, meiotic defects, and poor growth due to accumulation of cells with a late S/G 2 content of DNA (Wallis et al., 1989;Gangloff et al., 1994Gangloff et al., , 1999Chakraverty et al., 2001). Similarly, deletion of top3 ϩ in Schizosaccharomyces pombe causes lethality due to chromosome missegregation and accumulation of DNA double-strand breaks (Goodwin et al., 1999;Maftahi et al., 1999;Oh et al., 2002;Win et al., 2004). Whereas lower eukaryotes generally contain only one type IA topoisomerase, human cells, like most vertebrates, possess at least two Top3 homologues, hTOPIII␣ and hTOPIII␤ (Hanai et al., 1996;Ng et al., 1999). In mice, mutation of TOP3␣ causes embryonic lethality (Li and Wang, 1998), whereas mutation of TOP3␤ causes a shortened life span (Kwan et al., 2003). Interestingly, in S. cerevisiae and S. pombe, deletion of SGS1 or rqh1 ϩ can largely suppress the phenotypes caused by deletion of TOP3 or top3 ϩ , respectively (Gangloff et al., 1994;Goodwin et al., 1999;Maftahi et al., 1999;Chakraverty et al., 2001). Because both SGS1 and rqh1 ϩ belong to the same family of proteins, the RecQ DNA helicases, this intriguing genetic interaction has led to the suggestion that type IA topoisomerases may be functionally associated with RecQ helicases. Indeed, both Sgs1 and Rqh1 physically interact with Top3 in their respective organisms (Gangloff et al., 1994;Bennett et al., 2000;Fricke et al., 2001;Onodera et al., 2002;Laursen et al., 2003;Ahmad and Stewart, 2005;Ui et al., 2005), and a close as...

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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Top3 Processes Recombination Intermediates and Modulates Checkpoint Activity after DNA Damage

Mankouri

Hickson

2006

MBoC

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“…Protein-protein interactions were detected by quantitative ␤-galactosidase activity for permeabilized cells and represent the averages of three independent experiments, with error bars indicating S.D. (37).…”

Section: Methodsmentioning

confidence: 99%

During Replication Stress, Non-Smc Element 5 (Nse5) Is Required for Smc5/6 Protein Complex Functionality at Stalled Forks

Bustard

Menolfi

Jeppsson

et al. 2012

Journal of Biological Chemistry

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Background: The Smc5/6 complex has six non-SMC elements, including Nse5. Results: Utilizing two mutants of NSE5, we separated Smc5 sumoylation from Smc5/6 complex function. Conclusion: Nse5 integrity is important for Smc5/6 complex stability, which in turn is essential for localization of the complex to stalled forks. Significance: Our results provide the first in vivo characterization of Nse5 for Smc5/6 complex function.

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“…However, Sgs1 also displays functions that do not appear to require Top3 and Rmi1. For example, Top3 and Rmi1 are not required for DNA end resection, although they serve a stimulatory role , and Sgs1 functions independently of Top3 in stimulating the Rad53 checkpoint kinase in response to HU-dependent replication fork stalling, though this checkpoint function does not require the helicase activity of Sgs1 (Bjergbaek et al 2005).…”

Section: Top3 and Rmi1 Act In Heteroduplex Rejectionmentioning

confidence: 99%

A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences inSaccharomyces cerevisiae

Chakraborty¹,

George²,

Lyndaker

et al. 2015

Genetics

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Single-strand annealing (SSA) is an important homologous recombination mechanism that repairs DNA double strand breaks (DSBs) occurring between closely spaced repeat sequences. During SSA, the DSB is acted upon by exonucleases to reveal complementary sequences that anneal and are then repaired through tail clipping, DNA synthesis, and ligation steps. In baker's yeast, the Msh DNA mismatch recognition complex and the Sgs1 helicase act to suppress SSA between divergent sequences by binding to mismatches present in heteroduplex DNA intermediates and triggering a DNA unwinding mechanism known as heteroduplex rejection. Using baker's yeast as a model, we have identified new factors and regulatory steps in heteroduplex rejection during SSA. First we showed that Top3-Rmi1, a topoisomerase complex that interacts with Sgs1, is required for heteroduplex rejection. Second, we found that the replication processivity clamp proliferating cell nuclear antigen (PCNA) is dispensable for heteroduplex rejection, but is important for repairing mismatches formed during SSA. Third, we showed that modest overexpression of Msh6 results in a significant increase in heteroduplex rejection; this increase is due to a compromise in Msh2-Msh3 function required for the clipping of 39 tails. Thus 39 tail clipping during SSA is a critical regulatory step in the repair vs. rejection decision; rejection is favored before the 39 tails are clipped. Unexpectedly, Msh6 overexpression, through interactions with PCNA, disrupted heteroduplex rejection between divergent sequences in another recombination substrate. These observations illustrate the delicate balance that exists between repair and replication factors to optimize genome stability.

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Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance

Cited by 136 publications

References 51 publications

Top3 Processes Recombination Intermediates and Modulates Checkpoint Activity after DNA Damage

Top3 Processes Recombination Intermediates and Modulates Checkpoint Activity after DNA Damage

During Replication Stress, Non-Smc Element 5 (Nse5) Is Required for Smc5/6 Protein Complex Functionality at Stalled Forks

A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences inSaccharomyces cerevisiae

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