Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1.

Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in Xenopus egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of FUN30, the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with MSH6 or MSH3 deletion but no significant increase with MSH2 deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction.

Shelterin promotes tethering of late replication origins to telomeres for replication-timing control.

DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere-binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere-like sequence near the origins. Here, we showed using a lacO/LacI-GFP system that Taz1-dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication-timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication-timing control and telomeric association of Taz1-dependent late origins, and this requirement was bypassed by a minishelterin Tpz1-Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin-mediated telomeric association of the origins at the onset of S phase.

Telomere-binding protein Taz1 controls global replication timing through its localization near late replication origins in fission yeast.

In eukaryotes, the replication of chromosome DNA is coordinated by a replication timing program that temporally regulates the firing of individual replication origins. However, the molecular mechanism underlying the program remains elusive. Here, we report that the telomere-binding protein Taz1 plays a crucial role in the control of replication timing in fission yeast. A DNA element located proximal to a late origin in the chromosome arm represses initiation from the origin in early S phase. Systematic deletion and substitution experiments demonstrated that two tandem telomeric repeats are essential for this repression. The telomeric repeats recruit Taz1, a counterpart of human TRF1 and TRF2, to the locus. Genome-wide analysis revealed that Taz1 regulates about half of chromosomal late origins, including those in subtelomeres. The Taz1-mediated mechanism prevents Dbf4-dependent kinase (DDK)-dependent Sld3 loading onto the origins. Our results demonstrate that the replication timing program in fission yeast uses the internal telomeric repeats and binding of Taz1.

Regulation of mitotic recombination between DNA repeats in centromeres.

Centromeres that are essential for faithful segregation of chromosomes consist of unique DNA repeats in many eukaryotes. Although recombination is under-represented around centromeres during meiosis, little is known about recombination between centromere repeats in mitotic cells. Here, we compared spontaneous recombination that occurs between ade6B/ade6X inverted repeats integrated at centromere 1 (cen1) or at a non-centromeric ura4 locus in fission yeast. Remarkably, distinct mechanisms of homologous recombination (HR) were observed in centromere and non-centromere regions. Rad51-dependent HR that requires Rad51, Rad54 and Rad52 was predominant in the centromere, whereas Rad51-independent HR that requires Rad52 also occurred in the arm region. Crossovers between inverted repeats (i.e. inversions) were under-represented in the centromere as compared to the arm region. While heterochromatin was dispensable, Mhf1/CENP-S, Mhf2/CENP-X histone-fold proteins and Fml1/FANCM helicase were required to suppress crossovers. Furthermore, Mhf1 and Fml1 were found to prevent gross chromosomal rearrangements mediated by centromere repeats. These data for the first time uncovered the regulation of mitotic recombination between DNA repeats in centromeres and its physiological role in maintaining genome integrity.

Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation.

Centromeres consist of DNA repeats in many eukaryotes. Non-allelic homologous recombination (HR) between them can result in gross chromosomal rearrangements (GCRs). In fission yeast, Rad51 suppresses isochromosome formation that occurs between inverted repeats in the centromere. However, how the HR enzyme prevents homology-mediated GCRs remains unclear. Here, we provide evidence that Rad51 with the aid of the Swi/Snf-type motor protein Rad54 promotes non-crossover recombination between centromere repeats to prevent isochromosome formation. Mutations in Rad51 and Rad54 epistatically increased the rates of isochromosome formation and chromosome loss. In sharp contrast, these mutations decreased gene conversion between inverted repeats in the centromere. Remarkably, analysis of recombinant DNAs revealed that rad51 and rad54 increase the proportion of crossovers. In the absence of Rad51, deletion of the structure-specific endonuclease Mus81 decreased both crossovers and isochromosomes, while the cdc27/pol32-D1 mutation, which impairs break-induced replication, did not. We propose that Rad51 and Rad54 promote non-crossover recombination between centromere repeats on the same chromatid, thereby suppressing crossover between non-allelic repeats on sister chromatids that leads to chromosomal rearrangements. Furthermore, we found that Rad51 and Rad54 are required for gene silencing in centromeres, suggesting that HR also plays a role in the structure and function of centromeres.

Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components.

The CMG complex composed of Mcm2-7, Cdc45 and GINS is postulated to be the eukaryotic replicative DNA helicase, whose activation requires sequential recruitment of replication proteins onto Mcm2-7. Current models suggest that Mcm10 is involved in assembly of the CMG complex, and in tethering of DNA polymerase α at replication forks. Here, we report that Mcm10 is required for origin DNA unwinding after association of the CMG components with replication origins in fission yeast. A combination of promoter shut-off and the auxin-inducible protein degradation (off-aid) system efficiently depleted cellular Mcm10 to <0.5% of the wild-type level. Depletion of Mcm10 did not affect origin loading of Mcm2-7, Cdc45 or GINS, but impaired recruitment of RPA and DNA polymerases. Mutations in a conserved zinc finger of Mcm10 abolished RPA loading after recruitment of Mcm10. These results show that Mcm10, together with the CMG components, plays a novel essential role in origin DNA unwinding through its zinc-finger function.

Meiotic nuclear movements in fission yeast are regulated by the transcription factor Mei4 downstream of a Cds1-dependent replication checkpoint pathway.

In meiosis, the fission yeast nucleus displays an elongated morphology, moving back and forth within the cell; these nuclear movements continue for approximately 2 h before meiotic nuclear divisions. Meiotic DNA replication occurs in an early phase of the nuclear movements and is followed by meiotic prophase. Here we report that in mutants deficient in meiotic DNA replication, the duration of nuclear movements is strikingly prolonged to four to 5 h. We found that this prolongation was caused by the Cds1-dependent replication checkpoint, which represses expression of the mei4(+) gene encoding a meiosis-specific transcription factor. In the absence of Mei4, nuclear movements persisted for more than 8 h. In contrast, overproduction of Mei4 accelerated termination of nuclear movements to approximately 30 min. These results show that Mei4 is involved in the termination of nuclear movements and that Mei4-mediated regulatory pathways link a DNA replication checkpoint to the termination of nuclear movements.

Abundance of prereplicative complexes (Pre-RCs) facilitates recombinational repair under replication stress in fission yeast.

Mcm2-7 complexes are loaded onto chromatin with the aid of Cdt1 and Cdc18/Cdc6 and form prereplicative complexes (pre-RCs) at multiple sites on each chromosome. Pre-RCs are essential for DNA replication and surviving replication stress. However, the mechanism by which pre-RCs contribute to surviving replication stress is largely unknown. Here, we isolated the fission yeast mcm6-S1 mutant that was hypersensitive to methyl methanesulfonate (MMS) and camptothecin (CPT), both of which cause forks to collapse. The mcm6-S1 mutation impaired the interaction with Cdt1 and decreased the binding of minichromosome maintenance (MCM) proteins to replication origins. Overexpression of Cdt1 restored MCM binding and suppressed the sensitivity to MMS and CPT, suggesting that the Cdt1-Mcm6 interaction is important for the assembly of pre-RCs and the repair of collapsed forks. MMS-induced Chk1 phosphorylation and Rad22/Rad52 focus formation occurred normally, whereas cells containing Rhp54/Rad54 foci, which are involved in DNA strand exchange and dissociation of the joint molecules, were increased. Remarkably, G(1) phase extension through deletion of an S phase cyclin, Cig2, as well as Cdt1 overexpression restored pre-RC assembly and suppressed Rhp54 accumulation. A cdc18 mutation also caused hypersensitivity to MMS and CPT and accumulation of Rhp54 foci. These data suggest that an abundance of pre-RCs facilitates a late step in the recombinational repair of collapsed forks in the following S phase.

Regulation of DNA replication by chromatin structures: accessibility and recruitment.

The initiation of DNA replication and the elongation of DNA strands take place in chromatin, a huge compound DNA-protein complex. Although the factors involved in the process of DNA replication have been largely elucidated, the underlying mechanisms that determine their behavior in the context of chromatin have only recently begun to be understood. It has been known that transcription is tightly regulated by the state of chromatin compaction, which governs the accessibility of DNA to trans-acting factors. This process is influenced by several determinants of chromatin structure, including intrinsic nucleosome positioning, the nucleosome remodeling complex, histone post-translational modifiers, and histone- and DNA-binding proteins. Growing evidence indicates that this concept is also applicable to the regulation of DNA replication. In addition, recent studies have demonstrated a distinctive mode of regulation. Some non-histone chromatin-binding proteins have been shown to interact physically with replication factors, thereby facilitating their recruitment at specific chromosomal loci. This type of regulation may allow control of local replication activity without affecting other chromosomal processes.

Rad51 suppresses gross chromosomal rearrangement at centromere in Schizosaccharomyces pombe.

Centromere that plays a pivotal role in chromosome segregation is composed of repetitive elements in many eukaryotes. Although chromosomal regions containing repeats are the hotspots of rearrangements, little is known about the stability of centromere repeats. Here, by using a minichromosome that has a complete set of centromere sequences, we have developed a fission yeast system to detect gross chromosomal rearrangements (GCRs) that occur spontaneously. Southern and comprehensive genome hybridization analyses of rearranged chromosomes show two types of GCRs: translocation between homologous chromosomes and formation of isochromosomes in which a chromosome arm is replaced by a copy of the other. Remarkably, all the examined isochromosomes contain the breakpoint in centromere repeats, showing that isochromosomes are produced by centromere rearrangement. Mutations in the Rad3 checkpoint kinase increase both types of GCRs. In contrast, the deletion of Rad51 recombinase preferentially elevates isochromosome formation. Chromatin immunoprecipitation analysis shows that Rad51 localizes at centromere around S phase. These data suggest that Rad51 suppresses rearrangements of centromere repeats that result in isochromosome formation.

Auxin-inducible protein depletion system in fission yeast.

BACKGROUND: Inducible inactivation of a protein is a powerful approach for analysis of its function within cells. Fission yeast is a useful model for studying the fundamental mechanisms such as chromosome maintenance and cell cycle. However, previously published strategies for protein-depletion are successful only for some proteins in some specific conditions and still do not achieve efficient depletion to cause acute phenotypes such as immediate cell cycle arrest. The aim of this work was to construct a useful and powerful protein-depletion system in Shizosaccaromyces pombe. RESULTS: We constructed an auxin-inducible degron (AID) system, which utilizes auxin-dependent poly-ubiquitination of Aux/IAA proteins by SCFTIR1 in plants, in fission yeast. Although expression of a plant F-box protein, TIR1, decreased Mcm4-aid, a component of the MCM complex essential for DNA replication tagged with Aux/IAA peptide, depletion did not result in an evident growth defect. We successfully improved degradation efficiency of Mcm4-aid by fusion of TIR1 with fission yeast Skp1, a conserved F-box-interacting component of SCF (improved-AID system; i-AID), and the cells showed severe defect in growth. The i-AID system induced degradation of Mcm4-aid in the chromatin-bound MCM complex as well as those in soluble fractions. The i-AID system in conjunction with transcription repression (off-AID system), we achieved more efficient depletion of other proteins including Pol1 and Cdc45, causing early S phase arrest. CONCLUSION: Improvement of the AID system allowed us to construct conditional null mutants of S. pombe. We propose that the off-AID system is the powerful method for in vivo protein-depletion in fission yeast.

Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks.

The minichromosome maintenance (MCM) helicase, composed of subunits Mcm2-7, is essential for the initiation and elongation phases of DNA replication. Even when DNA synthesis is blocked, MCM continues DNA unwinding to some extent for activation of the replication checkpoint and then stops. However, the mechanism of regulation of MCM-helicase activity remains unknown. Here, we show that truncation of the Mcm4 C-terminal domain (CTD) in fission yeast results in hypersensitivity to replication block caused by dNTP depletion. The truncation mcm4-c84 does not affect the activation of the replication checkpoint pathway but delays its attenuation during recovery from replication block. Two dimensional gel electrophoresis showed that mcm4-c84 delays the disappearance of replication intermediates, indicating that the Mcm4 CTD is required for efficient recovery of stalled replication forks. Remarkably, chromatin immunoprecipitation revealed that mcm4-c84 brings about an increase rather than a decrease in the association of the single-stranded DNA-binding protein RPA to stalled forks, and MCM and the accessory complex GINS are unaffected. These results suggest that the Mcm4 CTD is required to suspend MCM-helicase activity after the formation of single-stranded DNA sufficient for checkpoint activation.

Heterochromatin suppresses gross chromosomal rearrangements at centromeres by repressing Tfs1/TFIIS-dependent transcription.

Heterochromatin, characterized by histone H3 lysine 9 (H3K9) methylation, assembles on repetitive regions including centromeres. Although centromeric heterochromatin is important for correct segregation of chromosomes, its exact role in maintaining centromere integrity remains elusive. Here, we found in fission yeast that heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres. Mutations in Clr4/Suv39 methyltransferase increased the formation of isochromosomes, whose breakpoints were located in centromere repeats. H3K9A and H3K9R mutations also increased GCRs, suggesting that Clr4 suppresses centromeric GCRs via H3K9 methylation. HP1 homologs Swi6 and Chp2 and the RNAi component Chp1 were the chromodomain proteins essential for full suppression of GCRs. Remarkably, mutations in RNA polymerase II (RNAPII) or Tfs1/TFIIS, the transcription factor that facilitates restart of RNAPII after backtracking, specifically bypassed the requirement of Clr4 for suppressing GCRs. These results demonstrate that heterochromatin suppresses GCRs by repressing Tfs1-dependent transcription of centromere repeats.

Regulation of DNA replication machinery by Mrc1 in fission yeast.

Faithful replication of chromosomes is crucial to genome integrity. In yeast, the ORC binds replication origins throughout the cell cycle. However, Cdc45 binds these before S-phase, and, during replication, it moves along the DNA with MCM helicase. When replication progression is inhibited, checkpoint regulation is believed to stabilize the replication fork; the detailed mechanism, however, remains unclear. To examine the relationship between replication initiation and elongation defects and the response to replication elongation block, we used fission yeast mutants of Orc1 and Cdc45--orp1-4 and sna41-928, respectively--at their respective semipermissive temperatures with regard to BrdU incorporation. Both orp1 and sna41 cells exhibited HU hypersensitivity in the absence of Chk1, a DNA damage checkpoint kinase, and were defective in full activation of Cds1, a replication checkpoint kinase, indicating that normal replication is required for Cds1 activation. Mrc1 is required to activate Cds1 and prevent the replication machinery from uncoupling from DNA synthesis. We observed that, while either the orp1 or the sna41 mutation partially suppressed HU sensitivity of cds1 cells, sna41 specifically suppressed that of mrc1 cells. Interestingly, sna41 alleviated the defect in recovery from HU arrest without increasing Cds1 activity. In addition to sna41, specific mutations of MCM suppressed the HU sensitivity of mrc1 cells. Thus, during elongation, Mrc1 may negatively regulate Cdc45 and MCM helicase to render stalled forks capable of resuming replication.

SpSld3 is required for loading and maintenance of SpCdc45 on chromatin in DNA replication in fission yeast.

Initiation of DNA replication in eukaryotic cells is regulated through the ordered assembly of replication complexes at origins of replication. Association of Cdc45 with the origins is a crucial step in assembly of the replication machinery, hence can be considered a target for the regulation of origin activation. To examine the process required for SpCdc45 loading, we isolated fission yeast SpSld3, a counterpart of budding yeast Sld3 that interacts with Cdc45. SpSld3 associates with the replication origin during G1-S phases and this association depends on Dbf4-dependent (DDK) kinase activity. In the corresponding period, SpSld3 interacts with minichromosome maintenance (MCM) proteins and then with SpCdc45. A temperature-sensitive sld3-10 mutation suppressed by the multicopy of the sna41+ encoding SpCdc45 impairs loading of SpCdc45 onto chromatin. In addition, this mutation leads to dissociation of preloaded Cdc45 from chromatin in the hydroxyurea-arrested S phase, and DNA replication upon removal of hydroxyurea is retarded. Thus, we conclude that SpSld3 is required for stable association of Cdc45 with chromatin both in initiation and elongation of DNA replication. The DDK-dependent origin association suggests that SpSld3 is involved in temporal regulation of origin firing.

A novel intermediate in initiation complex assembly for fission yeast DNA replication.

Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of preinitiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+). The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably because of a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast.

A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11(+) gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and gamma-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.

MutSα maintains the mismatch repair capability by inhibiting PCNA unloading.

Eukaryotic mismatch repair (MMR) utilizes single-strand breaks as signals to target the strand to be repaired. DNA-bound PCNA is also presumed to direct MMR. The MMR capability must be limited to a post-replicative temporal window during which the signals are available. However, both identity of the signal(s) involved in the retention of this temporal window and the mechanism that maintains the MMR capability after DNA synthesis remain unclear. Using Xenopus egg extracts, we discovered a mechanism that ensures long-term retention of the MMR capability. We show that DNA-bound PCNA induces strand-specific MMR in the absence of strand discontinuities. Strikingly, MutSα inhibited PCNA unloading through its PCNA-interacting motif, thereby extending significantly the temporal window permissive to strand-specific MMR. Our data identify DNA-bound PCNA as the signal that enables strand discrimination after the disappearance of strand discontinuities, and uncover a novel role of MutSα in the retention of the post-replicative MMR capability.

Shugoshin forms a specialized chromatin domain at subtelomeres that regulates transcription and replication timing.

A chromosome is composed of structurally and functionally distinct domains. However, the molecular mechanisms underlying the formation of chromatin structure and the function of subtelomeres, the telomere-adjacent regions, remain obscure. Here we report the roles of the conserved centromeric protein Shugoshin 2 (Sgo2) in defining chromatin structure and functions of the subtelomeres in the fission yeast Schizosaccharomyces pombe. We show that Sgo2 localizes at the subtelomeres preferentially during G2 phase and is essential for the formation of a highly condensed subtelomeric chromatin body 'knob'. Furthermore, the absence of Sgo2 leads to the derepression of the subtelomeric genes and premature DNA replication at the subtelomeric late origins. Thus, the subtelomeric specialized chromatin domain organized by Sgo2 represses both transcription and replication to ensure proper gene expression and replication timing.