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Centromere proteins CENP-S and CENP-X are members of the constitutive centromere-associated network, which is a conserved group of proteins that are needed for the assembly and function of kinetochores at centromeres. Intriguingly CENP-S and CENP-X have alter egos going by the names of MHF1 (FANCM-associated histone-fold protein 1) and MHF2 respectively. In this guise they function with a DNA translocase called FANCM (Fanconi's anemia complementation group M) to promote DNA repair and homologous recombination. In the present review we discuss current knowledge of the biological roles of CENP-S and CENP-X and how their dual existence may be a common feature of CCAN (constitutive centromere-associated network) proteins. more...

The Fanconi anemia (FA) core complex promotes the tolerance/repair of DNA damage at stalled replication forks by catalyzing the monoubiquitination of FANCD2 and FANCI. Intriguingly, the core complex component FANCM also catalyzes branch migration of model Holliday junctions and replication forks in vitro. Here we have characterized the ortholog of FANCM in fission yeast Fml1 in order to understand the physiological significance of this activity. We show that Fml1 has at least two roles in homologous recombination-it promotes Rad51-dependent gene conversion at stalled/blocked replication forks and limits crossing over during mitotic double-strand break repair. In vitro Fml1 catalyzes both replication fork reversal and D loop disruption, indicating possible mechanisms by which it can fulfill its pro- and antirecombinogenic roles. more...

Cells of all living organisms have evolved complex mechanisms that serve to stabilise, repair and restart stalled, blocked and broken replication forks. The heterodimeric Mus81-Eme1/Mms4 structure-specific endonuclease appears to play an important role(s) in homologous recombination-mediated processing of such perturbed forks. This enzyme has been implicated in the cleavage of stalled and blocked replication forks to initiate recombination, as well as in the processing of recombination intermediates that result from repairing damaged forks. In this review we assess the biochemical and genetic evidence for the mitotic role of Mus81-Eme1/Mms4 at replication forks and in repairing post-replication DNA damage. Mus81 appears to act when replication is impeded by genotoxins or by impairment of the replication machinery, or when arrested replication forks are not adequately protected. We discuss how its action is regulated by the S-phase cell cycle checkpoint, depending on the nature of the stalled or damaged fork. We also present a new way in which Mus81 may limit crossing over during the repair of post-replication gaps, and explore Mus81's interplay with other components of the recombination machinery, including the RecQ helicases that also play important roles in processing replication and recombination intermediates. more...

In budding yeast most Rad51-dependent and -independent recombination depends on Rad52. In contrast, its homologue in fission yeast, Rad22, was assumed to play a less critical role possibly due to functional redundancy with another Rad52-like protein Rti1. We show here that this is not the case. Rad22 like Rad52 plays a central role in recombination being required for both Rhp51-dependent and -independent events. Having established this we proceed to investigate the involvement of the Mus81-Eme1 endonuclease in these pathways. Mus81 plays a relatively minor role in the Rhp51-dependent repair of DNA damage induced by ultraviolet light. In contrast Mus81 has a key role in the Rad22-dependent (Rhp51-independent) repair of damage induced by camptothecin, hydroxyurea and methyl-methanesulfonate. Furthermore, spontaneous intrachromosomal recombination that gives rise to deletion recombinants is impaired in a mus81 mutant. From these data we propose that a Rad22-Mus81-dependent (Rhp51-independent) pathway is an important mechanism for the repair of DNA damage in fission yeast. Consistent with this we show that in vitro Rad22 can promote strand invasion to form a D-loop that can be cleaved by Mus81. more...

One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3'-blocking termini following AP lyase cleavage by Nth1. more...

Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage. Cell cycle delays in response to DNA damage are mediated via checkpoint proteins. Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G(2)/M checkpoint stops cells passing from G(2) into mitosis. In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G(2)/M checkpoint. Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks. Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels. Mutants lacking the G(2)/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants. This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G(2)/M mutants after UV irradiation. Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G(2)/M checkpoint mutants. more...

Summary: Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or γ-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [[sup 3]H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.. [ABSTRACT FROM AUTHOR] more...