Abstract Dynamic changes in chromosomal structure that occur during meiotic prophase play an important role in the progression of meiosis. Among them, meiosis‐specific chromosomal axis‐loop structures are important as a scaffold for integrated control between the meiotic recombination reaction and the associated checkpoint system to ensure accurate chromosome segregation. However, the molecular mechanism of the initial step of chromosome axis‐loop construction is not well understood. Here, we showed that, in budding yeast, protein phosphatase 4 (PP4) that primarily counteracts Mec1/Tel1 phosphorylation is required to promote the assembly of a chromosomal axis component Hop1 and Red1 onto meiotic chromatin via interaction with Hop1. PP4, on the other hand, less affects Rec8 assembly. Notably, unlike the previously known function of PP4, this PP4 function in Hop1/Red1 assembly was independent of meiotic DSB‐dependent Tel1/Mec1 kinase activities. The defect in Hop1/Red1 assembly in the absence of PP4 function was not suppressed by dysfunction of Pch2, which removes Hop1 protein from the chromosome axis, suggesting that PP4 is required for the initial step of chromatin loading of Hop1 rather than stabilization of Hop1 on axes. These results indicate phosphorylation/dephosphorylation‐mediated regulation of Hop1 recruitment onto chromatin during chromosome axis construction before meiotic double‐strand break formation.

Meiotic crossing over is essential for the segregation of homologous chromosomes. The formation and distribution of meiotic crossovers (COs), which are initiated by the formation of double-strand break (DSB), are tightly regulated to ensure at least one CO per bivalent. One type of CO control, CO homeostasis, maintains a consistent level of COs despite fluctuations in DSB numbers. Here, we analyzed the localization of proteins involved in meiotic recombination in budding yeast xrs2 hypomorphic mutants which show different levels of DSBs. The number of cytological foci with recombinases, Rad51 and Dmc1, which mark single-stranded DNAs at DSB sites is proportional to the DSB numbers. Among the pro-CO factor, ZMM/SIC proteins, the focus number of Zip3, Mer3, or Spo22/Zip4, was linearly proportional to reduced DSBs in the xrs2 mutant. In contrast, foci of Msh5, a component of the MutSγ complex, showed a non-linear response to reduced DSBs. We also confirmed the homeostatic response of COs by genetic analysis of meiotic recombination in the xrs2 mutants and found a chromosome-specific homeostatic response of COs. Our study suggests that the homeostatic response of the Msh5 assembly to reduced DSBs was genetically distinct from that of the Zip3 assembly for CO control.

Abstract Acetaldehyde, a metabolic product of ethanol, induces DNA damage and genome instability. Accumulation of acetaldehyde due to alcohol consumption or aldehyde dehydrogenase (ALDH2) deficiency increases the risks of various types of cancers, including esophageal cancer. Although acetaldehyde chemically induces DNA adducts, the repair process of the lesions remains unclear. To investigate the mechanism of repair of acetaldehyde-induced DNA damage, we determined the repair pathway using siRNA knockdown and immunofluorescence assays of repair factors. Herein, we report that acetaldehyde induces DNA double-strand breaks (DSBs) in human U2OS cells and that both DSB repair pathways, non-homologous end-joining (NHEJ) and homology-directed repair (HDR), are required for the repair of acetaldehyde-induced DNA damage. Our findings suggest that acetaldehyde-induced DNA adducts are converted into DSBs and repaired via NHEJ or HDR in human cells. To reduce the risk of acetaldehyde-associated carcinogenesis, we investigated potential strategies of reducing acetaldehyde-induced DNA damage. We report that polyphenols extracted from persimmon fruits and epigallocatechin, a major component of persimmon polyphenols, attenuate acetaldehyde-induced DNA damage without affecting the repair kinetics. The data suggest that persimmon polyphenols suppress DSB formation by scavenging acetaldehyde. Persimmon polyphenols can potentially inhibit carcinogenesis following alcohol consumption.

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.

Abstract In the baker’s yeast Saccharomyces cerevisiae, most of the meiotic crossovers are generated through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex. To understand the role of Msh4-Msh5 in meiotic crossing over, we determined its genome wide in vivo binding sites in meiotic cells. We show that Msh5 specifically associates with DSB hotspots, chromosome axes, and centromeres on chromosomes. A basal level of Msh5 association with these chromosomal features is observed even in the absence of DSB formation (spo11Δ mutant) at the early stages of meiosis. But efficient binding to DSB hotspots and chromosome axes requires DSB formation and resection and is enhanced by double Holliday junction structures. Msh5 binding is also correlated to DSB frequency and enhanced on small chromosomes with higher DSB and crossover density. The axis protein Red1 is required for Msh5 association with the chromosome axes and DSB hotspots but not centromeres. Although binding sites of Msh5 and other pro-crossover factors like Zip3 show extensive overlap, Msh5 associates with centromeres independent of Zip3. These results on Msh5 localization in wild type and meiotic mutants have implications for how Msh4-Msh5 works with other pro-crossover factors to ensure crossover formation.

The synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. In budding yeast, Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. During SC elongation, the SUMOylated SC component Ecm11 and the Ecm11-interacting protein Gmc2 facilitate the polymerization of Zip1, an SC central region component. Through physical recombination, cytological, and genetic analyses, we found that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies on a short chromosome (chromosome III) were reduced, whereas CO and non-crossover frequencies on a long chromosome (chromosome VII) were elevated. Further, in ecm11 and gmc2 mutants, more double-strand breaks (DSBs) were formed on a long chromosome during late prophase I, implying that the Ecm11-Gmc2 (EG) complex is involved in the homeostatic regulation of DSB formation. The EG complex may participate in joint molecule (JM) processing and/or double-Holliday junction resolution for ZMM-dependent CO-designated recombination. Absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role for the EG complex in suppressing ZMM-independent recombination. Our results suggest that the SC central region functions as a compartment for sequestering recombination-associated proteins to regulate meiosis specificity during recombination.

Homologous chromosomes pair with each other during meiosis, culminating in the formation of the synaptonemal complex (SC), which is coupled with meiotic recombination. In this study, we showed that a meiosis-specific depletion mutant of a cullin (Cdc53) in the SCF (Skp-Cullin-F-box) ubiquitin ligase, which plays a critical role in cell cycle regulation during mitosis, is deficient in SC formation. However, the mutant is proficient in forming crossovers, indicating the uncoupling of meiotic recombination with SC formation in the mutant. Furthermore, the deletion of the PCH2 gene encoding a meiosis-specific AAA+ ATPase suppresses SC-assembly defects induced by CDC53 depletion. On the other hand, the pch2 cdc53 double mutant is defective in meiotic crossover formation, suggesting the assembly of SC with unrepaired DNA double-strand breaks. A temperature-sensitive mutant of CDC4, which encodes an F-box protein of SCF, shows meiotic defects similar to those of the CDC53-depletion mutant. These results suggest that SCFCdc4, probably SCFCdc4-dependent protein ubiquitylation, regulates and collaborates with Pch2 in SC assembly and meiotic recombination.

Homologous recombination is essential for chromosome segregation during meiosis I. Meiotic recombination is initiated by the introduction of double-strand breaks (DSBs) at specific genomic locations called hotspots, which are catalyzed by Spo11 and its partners. DSB hotspots during meiosis are marked with Set1-mediated histone H3K4 methylation. The Spo11 partner complex, Rec114-Mer2-Mei4, essential for the DSB formation, localizes to the chromosome axes. For efficient DSB formation, a hotspot with histone H3K4 methylation on the chromatin loops is tethered to the chromosome axis through the H3K4 methylation reader protein, Spp1, on the axes, which interacts with Mer2. In this study, we found genetic interaction of mutants in a histone modification protein complex called PAF1C with the REC114 and MER2 in the DSB formation in budding yeast Saccharomyces cerevisiae. Namely, the paf1c mutations rtf1 and cdc73 showed synthetic defects in meiotic DSB formation only when combined with a wild-type-like tagged allele of either the REC114 or MER2. The synthetic defect of the tagged REC114 allele in the DSB formation was seen also with the set1, but not with spp1 deletion. These results suggest a novel role of histone modification machinery in DSB formation during meiosis, which is independent of Spp1-mediated loop-axis tethering.

The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.

The number and distribution of meiotic crossovers (COs) are highly regulated, reflecting the requirement for COs during the first round of meiotic chromosome segregation. CO control includes CO assurance and CO interference, which promote at least one CO per chromosome bivalent and evenly-spaced COs, respectively. Previous studies revealed a role for the DNA damage response (DDR) clamp and the clamp loader in CO formation by promoting interfering COs and interhomolog recombination, and also by suppressing ectopic recombination. In this study, we use classical tetrad analysis of Saccharomyces cerevisiae to show that a mutant defective in , which encodes the DDR clamp loader ( in other organisms), displayed reduced CO frequencies on two shorter chromosomes (III and V), but not on a long chromosome (chromosome VII). The residual COs in the mutant do not show interference. In contrast to , mutants defective in the ATR kinase homolog , including a null and a kinase-dead mutant, show slight or few defects in CO frequency. On the other hand, COs show defects in interference, similar to the mutant. Our results support a model in which the DDR clamp and clamp-loader proteins promote interfering COs by recruiting pro-CO Zip, Mer, and Msh proteins to recombination sites, while the kinase regulates CO distribution by a distinct mechanism. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.

Sister chromatid cohesion is essential for chromosome segregation both in mitosis and meiosis. Cohesion between two chromatids is mediated by a protein complex called cohesin. The loading and unloading of the cohesin are tightly regulated during the cell cycle. In vertebrate cells, cohesin is released from chromosomes by two distinct pathways. The best characterized pathway occurs at the onset of anaphase, when the kleisin component of the cohesin is destroyed by a protease, separase. The cleavage of the cohesin by separase releases entrapped sister chromatids allowing anaphase to commence. In addition, prior to the metaphase-anaphase transition, most of cohesin is removed from chromosomes in a cleavage-independent manner. This cohesin release is referred to as the prophase pathway. In meiotic cells, sister chromatid cohesion is essential for the segregation of homologous chromosomes during meiosis I. Thus, it was assumed that the prophase pathway for cohesin removal from chromosome arms would be suppressed during meiosis to avoid errors in chromosome segregation. However, recent studies revealed the presence of a meiosis-specific prophase-like pathway for cleavage-independent removal of cohesin during late prophase I in different organisms. In budding yeast, the cleavage-independent removal of cohesin is mediated through meiosis-specific phosphorylation of cohesin subunits, Rec8, the meiosis-specific kleisin, and the yeast Wapl ortholog, Rad61/Wpl1. This pathway plays a role in chromosome morphogenesis during late prophase I, promoting chromosome compaction. In this review, we give an overview of the prophase pathway for cohesin dynamics during meiosis, which has a complex regulation leading to differentially localized populations of cohesin along meiotic chromosomes.

Sister chromatid cohesion on chromosome arms is essential for the segregation of homologous chromosomes during meiosis I while it is dispensable for sister chromatid separation during mitosis. It was assumed that, unlike the situation in mitosis, chromosome arms retain cohesion prior to onset of anaphase-I. Paradoxically, reduced immunostaining signals of meiosis-specific cohesin, including the kleisin Rec8, were observed on chromosomes during late prophase-I of budding yeast. This decrease is seen in the absence of Rec8 cleavage and depends on condensin-mediated recruitment of Polo-like kinase (PLK/Cdc5). In this study, we confirmed that this release indeed accompanies the dissociation of acetylated Smc3 as well as Rec8 from meiotic chromosomes during late prophase-I. This release requires, in addition to PLK, the cohesin regulator, Wapl (Rad61/Wpl1 in yeast), and Dbf4-dependent Cdc7 kinase (DDK). Meiosis-specific phosphorylation of Rad61/Wpl1 and Rec8 by PLK and DDK collaboratively promote this release. This process is similar to the vertebrate prophase pathway for cohesin release during G2 phase and pro-metaphase. In yeast, meiotic cohesin release coincides with PLK-dependent compaction of chromosomes in late meiotic prophase-I. We suggest that yeast uses this highly regulated cleavage-independent pathway to remove cohesin during late prophase-I to facilitate morphogenesis of condensed metaphase-I chromosomes.Author summary In meiosis the life and health of future generations is decided upon. Any failure in chromosome segregation has a detrimental impact. Therefore, it is currently believed that the physical connections between homologous chromosomes are maintained by meiotic cohesin with exceptional stability. Indeed, it was shown that cohesive cohesin does not show an appreciable turnover during long periods in oocyte development. In this context, it was long assumed but not properly investigated, that the prophase pathway for cohesin release would be specific to mitosis and would be safely suppressed during meiosis so as not to endanger essential connections between chromosomes. However, a previous study on budding yeast meiosis suggests the presence of cleavage-independent pathway of cohesin release during late prophase-I. In the work presented here we confirmed that the prophase pathway is not suppressed during meiosis, at least in budding yeast and showed that this cleavage-independent release is regulated by meiosis-specific phosphorylation of two cohesin subunits, Rec8 and Rad61(Wapl) by two cell-cycle regulators, PLK and DDK. Our results suggest that late meiotic prophase-I actively controls cohesin dynamics on meiotic chromosomes for chromosome segregation.

Proteins in the nuclear envelope (NE) play a role in the dynamics and functions of the nucleus and of chromosomes during mitosis and meiosis. Mps3, a yeast NE protein with a conserved SUN domain, predominantly localizes on a yeast centrosome equivalent, spindle pole body (SPB), in mitotic cells. During meiosis, Mps3, together with SPB, forms a distinct multiple ensemble on NE. How meiosis-specific NE localization of Mps3 is regulated remains largely unknown. In this study, we found that a meiosis-specific component of the protein complex essential for sister chromatid cohesion, Rec8, binds to Mps3 during meiosis and controls Mps3 localization and proper dynamics on NE. Ectopic expression of Rec8 in mitotic yeast cells induced the formation of Mps3 patches/foci on NE. This required the cohesin regulator, WAPL ortholog, Rad61/Wpl1, suggesting that a meiosis-specific cohesin complex with Rec8 controls NE localization of Mps3. We also observed that two domains of the nucleoplasmic region of Mps3 are essential for NE localization of Mps3 in mitotic as well as meiotic cells. We speculate that the interaction of Mps3 with the meiosis-specific cohesin in the nucleoplasm is a key determinant for NE localization/function of Mps3.

A DNA double strand break (DSB) is one of the most cytotoxic DNA lesions, but it can be repaired by non homologous end joining (NHEJ) or by homologous recombination. The choice between these two repair pathways depends on the cell cycle stage. Although NHEJ constitutes a simple re-ligation reaction, the regulatory mechanism(s) controlling its activity has not been fully characterized. Lif1 is a regulatory subunit of the NHEJ-specific DNA ligase IV and interacts with Xrs2 of the MRX complex which is a key factor in DSB repair. Specifically, the C-terminal region of Lif1, which contains a CK2-specific phosphorylation motif, interacts with the FHA domain of Xrs2 during canonical-NHEJ (C-NHEJ). Herein, we show that Lif1 and Cka2, a catalytic subunit of yeast CK2, interact and that the C-terminal phosphorylation consensus motif in Lift is phosphorylated by recombinant CK2. These observations suggest that phosphorylation of Lift by CK2 at a DSB site promotes the Lif1-Xrs2 interaction and facilitates C-NHEJ. (C) 2018 The Authors. Published by Elsevier Inc.

The malaria parasite Plasmodium falciparum proliferates in the blood stream where the host immune system is most active. To escape from host immunity, P. falciparum has developed a number of evasion mechanisms. Serine repeat antigen 5 (SERA5) is a blood stage antigen highly expressed at late trophozoite and schizont stages. The P47 N-terminal domain of SERA5, the basis of SE36 antigen of the blood stage vaccine candidate under clinical trials, covers the merozoite surface. Exploring the role of the P47 domain, screening of serum proteins showed that vitronectin (VTN) directly binds to 20 residues in the C-terminal region of SE36. VTN co-localized with P47 domain in the schizont and merozoite stages. Phagocytosis assay using THP-1 cells demonstrated that VTN bound to SE36 prevented engulfment of SE36-beads. In addition, several serum proteins localized on the merozoite surface, suggesting that host proteins camouflage merozoites against host immunity via binding to VTN.

The number and distribution of meiosis crossover (CO) events on each bivalent are strictly controlled by multiple mechanisms to assure proper chromosome segregation during the first meiotic division. In Saccharomyces cerevisiae, Slx4 is a multi-functional scaffold protein for structure-selective endonucleases, such as Slx1 and Rad1 (which are involved in DNA damage repair), and is also a negative regulator of the Rad9-dependent signaling pathway with Rtt107. Slx4 has been believed to play only a minor role in meiotic recombination. Here, we report that Slx4 is involved in proper intrachromosomal distribution of meiotic CO formation, especially in regions near centromeres. We observed an increase in uncontrolled CO formation only in a region near the centromere in the slx4 Delta mutant. Interestingly, this phenomenon was not observed in the slx1 Delta, rad1 Delta, or rtt107 Delta mutants. In addition, we observed a reduced number of DNA double-strand breaks (DSBs) and altered meiotic DSB distribution on chromosomes in the slx4 Delta mutant. This suggests that the multi-functional Slx4 is required for proper CO formation and meiotic DSB formation.

Because DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR) or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA) domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.

Faithful meiotic chromosome segregation and fertility require meiotic recombination between homologous chromosomes rather than the equally available sister chromatid, a bias that in Saccharomyces cerevisiae depends on the meiotic kinase, Mek1. Mek1 is thought to mediate repair template bias by specifically suppressing sister-directed repair. Instead, we found that when Mek1 persists on closely paired ( synapsed) homologues, DNA repair is severely delayed, suggesting that Mek1 suppresses any proximal repair template. Accordingly, Mek1 is excluded from synapsed homologues in wild-type cells. Exclusion requires the AAA(+)-ATPase Pch2 and is directly coupled to synaptonemal complex assembly. Stage-specific depletion experiments further demonstrate that DNA repair in the context of synapsed homologues requires Rad54, a repair factor inhibited by Mek1. These data indicate that the sister template is distinguished from the homologue primarily by its closer proximity to inhibitory Mek1 activity. We propose that once pairing or synapsis juxtaposes homologues, exclusion of Mek1 is necessary to avoid suppression of all templates and accelerate repair progression.

Histone modification is a critical determinant of the frequency and location of meiotic double-strand breaks (DSBs), and thus recombination. Set1-dependent histone H3K4 methylation and Dot1-dependent H3K79 methylation play important roles in this process in budding yeast. Given that the RNA polymerase II associated factor 1 complex, Paf1C, promotes both types of methylation, we addressed the role of the Paf1C component, Rtf1, in the regulation of meiotic DSB formation. Similar to a set1 mutation, disruption of RTF1 decreased the occurrence of DSBs in the genome. However, the rtf1 set1 double mutant exhibited a larger reduction in the levels of DSBs than either of the single mutants, indicating independent contributions of Rtf1 and Set1 to DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a manner that was different from the distributions observed in both set1 and set1 dot1 mutants, including enhanced DSB formation at some DSB-cold regions that are occupied by nucleosomes in wild-type cells. These observations suggest that Rtf1, and by extension the Paf1C, modulate the genomic DSB landscape independently of H3K4 methylation.

Meiosis-specific cohesin, required for the linking of the sister chromatids, plays a critical role in various chromosomal events during meiotic prophase I, such as chromosome morphogenesis and dynamics, as well as recombination. Rad61/Wpl1 (Wapl in other organisms) negatively regulates cohesin functions. In this study, we show that meiotic chromosome axes are shortened in the budding yeast rad61/wpl1 mutant, suggesting that Rad61/Wpl1 negatively regulates chromosome axis compaction. Rad61/Wpl1 is required for efficient resolution of telomere clustering during meiosis I, indicating a positive effect of Rad61/Wpl1 on the cohesin function required for telomere dynamics. Additionally, we demonstrate distinct activities of Rad61/Wpl1 during the meiotic recombination, including its effects on the efficient processing of intermediates. Thus, Rad61/Wpl1 both positively and negatively regulates various cohesin-mediated chromosomal processes during meiosis.

Formation of crossovers between homologous chromosomes during meiosis is positively regulated by the ZMM proteins (also known as SIC proteins). DNA damage checkpoint proteins also promote efficient formation of interhomolog crossovers. Here, we examined, in budding yeast, the meiotic role of the heterotrimeric DNA damage response clamp composed of Rad17, Ddc1 and Mec3 (known as '9-1-1' in other organisms) and a component of the clamp loader, Rad24 (known as Rad17 in other organisms). Cytological analysis indicated that the 9-1-1 clamp and its loader are not required for the chromosomal loading of RecA homologs Rad51 or Dmc1, but are necessary for the efficient loading of ZMM proteins. Interestingly, the loading of ZMM proteins onto meiotic chromosomes was independent of the checkpoint kinase Mec1 (the homolog of ATR) as well as Rad51. Furthermore, the ZMM member Zip3 (also known as Cst9) bound to the 9-1-1 complex in a cell-free system. These data suggest that, in addition to promoting interhomolog bias mediated by Rad51-Dmc1, the 9-1-1 clamp promotes crossover formation through a specific role in the assembly of ZMM proteins. Thus, the 9-1-1 complex functions to promote two crucial meiotic recombination processes, the regulation of interhomolog recombination and crossover formation mediated by ZMM.

Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G(1) phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.

Epigenetic marks such as histone modifications play roles in various chromosome dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and K79 is involved in the initiation of recombination and the recombination checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes as well as methylation-defective mutants of histone H3. We confirmed the role of Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs) in meiosis for the initiation of meiotic recombination, and we showed the involvement of Dot1 (H3K79 methylation) in DSB formation in the absence of Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4 methylation-defective mutants are defective in SC elongation, although they seem to have moderate reduction of DSBs. This suggests that high levels of DSBs mediated by histone H3K4 methylation promote SC elongation.

Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions. © 2013 Elsevier Inc.

Homologous recombination is associated with the dynamic assembly and disassembly of DNA-protein complexes. Assembly of a nucleoprotein filament comprising ssDNA and the RecA homolog, Rad51, is a key step required for homology search during recombination. The budding yeast Srs2 DNA translocase is known to dismantle Rad51 filament in vitro. However, there is limited evidence to support the dismantling activity of Srs2 in vivo. Here, we show that Srs2 indeed disrupts Rad51-containing complexes from chromosomes during meiosis. Overexpression of Srs2 during the meiotic prophase impairs meiotic recombination and removes Rad51 from meiotic chromosomes. This dismantling activity is specific for Rad51, as Srs2 Overexpression does not remove Dmc1 (a meiosis-specific Rad51 homolog), Rad52 (a Rad51 mediator), or replication protein A (RPA; a single-stranded DNA-binding protein). Rather, RPA replaces Rad51 under these conditions. A mutant Srs2 lacking helicase activity cannot remove Rad51 from meiotic chromosomes. Interestingly, the Rad51-binding domain of Srs2, which is critical for Rad51-dismantling activity in vitro, is not essential for this activity in vivo. Our results suggest that a precise level of Srs2, in the form of the Srs2 translocase, is required to appropriately regulate the Rad51 nucleoprotein filament dynamics during meiosis.

During homologous recombination, eukaryotic RecA homologue Rad51 assembles into a nucleoprotein filament on single-stranded DNA to catalyse homologous pairing and DNA-strand exchange with a homologous template. Rad51 nucleoprotein filaments are highly dynamic and regulated via the coordinated actions of various accessory proteins including Rad51 mediators. Here, we identify a new Rad51 mediator complex. The PCSS complex, comprising budding yeast Psy3, Csm2, Shu1 and Shu2 proteins, binds to recombination sites and is required for Rad51 assembly and function during meiosis. Within the hetero-tetramer, Psy3-Csm2 constitutes a core sub-complex with DNA-binding activity. In vitro, purified Psy3-Csm2 stabilizes the Rad51-single-stranded DNA complex independently of nucleotide cofactor. The mechanism of Rad51 stabilization is inferred by our high-resolution crystal structure, which reveals Psy3-Csm2 to be a structural mimic of the Rad51-dimer, a fundamental unit of the Rad51-filament. Together, these results reveal a novel molecular mechanism for this class of Rad51-mediators, which includes the human Rad51 paralogues.

Recombination during meiosis in the form of crossover events promotes the segregation of homologous chromosomes by providing the only physical linkage between these chromosomes. Recombination occurs not only between allelic sites but also between non-allelic (ectopic) sites. Ectopic recombination is often suppressed to prevent non-productive linkages. In this study, we examined the effects of various mutations in genes involved in meiotic recombination on ectopic recombination during meiosis. RAD24, a DNA damage checkpoint clamp-loader gene, suppressed ectopic recombination in wild type in the same pathway as RAD51. In the absence of RAD24, a meiosis-specific recA homolog, DMC1, suppressed the recombination. Homology search and strand exchange in ectopic recombination occurred when either the RAD51 or the DMC1 recA homolog was absent, but was promoted by RAD52. Unexpectedly, the zip1 mutant, which is defective in chromosome synapsis, showed a decrease, rather than an increase, in ectopic recombination. Our results provide evidence for two types of ectopic recombination: one that occurs in wild-type cells and a second that occurs predominantly when the checkpoint pathway is inactivated.

DNA double-strand breaks (DSBs) are repaired by two distinct pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). NHEJ includes two pathways, that is, precise and imprecise end joining. We found that Lif1, a component of the DNA ligase IV complex in Saccharomyces cerevisiae, was phosphorylated by cyclin-dependent kinase (CDK) at Ser261 during the S to G2 phase but not during G1 phase. This phosphorylation was required for efficient NHEJ in G2/M cells, rather than in G1 cells. It also promotes the stable binding of Lif1 protein to DSBs, specifically in G2/M-arrested cells, which shows the resection of DSB ends. Thus, Lif1 phosphorylation plays a critical role in a certain type of imprecise NHEJ accompanied by DSB end resection and micro-homology. Lif1 phosphorylation at Ser261 is probably involved in micro-homology-dependent end joining associated with producing single-stranded DSB ends that are formed by Sae2 as early intermediates in the HR pathway. CDK-dependent modification of the NHEJ pathway might make DSB ends compatible for NHEJ and thus prevent competition between HR and NHEJ in hierarchy on the choice of DSB repair pathways.

In budding yeast, Mps3 is essential for duplicating the spindle pole body (SPB) and is critical for promoting chromosome motion during meiosis. It is a member of the SUN (Sad1-Unc-84) domain family of proteins that localizes to the inner nuclear envelope (NE) in many eukaryotic organisms and preferentially localizes to the SPB in vegetative growth; in meiotic prophase I, it redistributes to many sites within the NE. We constructed an mps3 mutant, mps3-sun, which completely lacks the SUN domain. Surprisingly, the mps3-sun mutation does not disrupt SPB duplication or Mps3 localization to the NE in meiosis. However, it confers several defects during meiotic prophase I including reduced chromosome motion, premature synapsis between homologous chromosomes, and reduced levels of closely juxtaposed homologous loci in pachytene. These findings suggest that in meiosis, the Mps3 SUN domain is important for modulating chromosome motion events that act in meiotic chromosome juxtaposition and by extension, promoting proper morphogenesis of the synaptonemal complex.

Cyclin-dependent protein kinases (CDKs) are required for various cell cycle events both in mitosis and in meiosis. During the meiotic prophase of Saccharomyces cerevisiae, only one CDK, Cdc28, which forms a complex with B-type cyclins, Clb5 or Clb6, promotes not only the onset of premeiotic DNA replication but also the formation of meiotic double-strand breaks (DSBs). In this study, we showed that Cdc28 exhibits punctate staining on chromosomes during meiotic prophase I. Chromosomal localization of Cdc28, dependent on Clb5 and/or Clb6, is frequently observed in zygotene and pachytene, when formation of the synaptonemal complex (SC) occurs. Interestingly, the CDK localization is independent of DSB formation, but rather dependent on meiosis-specific chromosome components such as Red1, Hop1 and a cohesin subunit Rec8. Compromised CDK activity in meiotic prophase leads to defective SC formation without affecting DSB formation. These results suggest that CDK-dependent phosphorylation regulates meiotic chromosome morphogenesis.

During meiosis, the Msh4-Msh5 complex is thought to stabilize single-end invasion intermediates that form during early stages of recombination and subsequently bind to Holliday junctions to facilitate crossover formation. To analyze Msh4-Msh5 function, we mutagenized 57 residues in Saccharomyces cerevisiae Msh4 and Msh5 that are either conserved across all Msh4/5 family members or are specific to Msh4 and Msh5. The Msh5 subunit appeared more sensitive to mutagenesis. We identified msh4 and msh5 threshold (msh4/5-t) mutants that showed wild-type spore viability and crossover interference but displayed, compared to wild-type, up to a two-fold decrease in crossing over on large and medium sized chromosomes (XV, VII, VIII). Crossing over on a small chromosome, however, approached wild-type levels. The msh4/5-t mutants also displayed synaptonemal complex assembly defects. A triple mutant containing a msh4/5-t allele and mutations that decreased meiotic double-strand break levels (spo11-HA) and crossover interference (pch2D) showed synergistic defects in spore viability. Together these results indicate that the baker's yeast meiotic cell does not require the,90 crossovers maintained by crossover homeostasis to form viable spores. They also show that Pch2-mediated crossover interference is important to maintain meiotic viability when crossovers become limiting.

During meiotic prophase, chromosomes display rapid movement, and their telomeres attach to the nuclear envelope and cluster to form a "chromosomal bouquet.'' Little is known about the roles of the chromosome movement and telomere clustering in this phase. In budding yeast, telomere clustering is promoted by a meiosis-specific, telomere-binding protein, Ndj1. Here, we show that a meiosis-specific protein, Csm4, which forms a complex with Ndj1, facilitates bouquet formation. In the absence of Csm4, Ndj1-bound telomeres tether to nuclear envelopes but do not cluster, suggesting that telomere clustering in the meiotic prophase consists of at least two distinct steps: Ndj1-dependent tethering to the nuclear envelope and Csm4-dependent clustering/movement. Similar to Ndj1, Csm4 is required for several distinct steps during meiotic recombination. Our results suggest that Csm4 promotes efficient second-end capture of a double-strand break following a homology search, as well as resolution of the double-Holliday junction during crossover formation. We propose that chromosome movement and associated telomere dynamics at the nuclear envelope promotes the completion of key biochemical steps during meiotic recombination.

Haploidization of the genome in meiosis requires that chromosomes be sorted exclusively into pairs stabilized by synaptonemal complexes (SCs) and crossovers. This sorting and pairing is accompanied by active chromosome positioning in meiotic prophase in which telomeres cluster near the spindle pole to form the bouquet before dispersing around the nuclear envelope. We now describe telomere-led rapid prophase movements (RPMs) that frequently exceed 1 mu m/s and persist throughout meiotic prophase. Bouquet formation and RPMs depend on NDJ1, MPS3, and a new member of this pathway, CSM4, which encodes a meiosis-specific nuclear envelope protein required specifically for telomere mobility. RPMs initiate independently of recombination but differ quantitatively in mutants that fail to complete recombination, suggesting that RPMs respond to recombination status. Together with recombination defects described for ndj1, our observations suggest that RPMs and SCs balance the disruption and stabilization of recombinational interactions, respectively, to regulate crossing over.

DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2 a homolog of human Nbs1, is a component of the Mre1 1-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but. is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dn14-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human.

Meiotic crossing-over is highly regulated such that each homolog pair typically receives at least one crossover (assurance) and adjacent crossovers are widely spaced (interference). Here we provide evidence that interference and assurance are mechanistically distinct processes that are separated by mutations in a new ZMM (Zip, Msh, Mer) protein from Saccharomyces cerevisiae, Spo16. Like other zmm mutants, spo16 cells have defects in both crossing-over and synaptonemal complex formation. Unlike in previously characterized zmm mutants, the residual crossovers in spo16 cells show interference comparable to that in the wild type. Spo16 interacts with a second ZMM protein, Spo22 (also known as Zip4), and spo22 mutants also show normal interference. Notably, assembly of the MutS homologs Msh4 and Msh5 on chromosomes occurs in both spo16 and spo22, but not in other zmm mutants. We suggest that crossover interference requires the normal assembly of recombination complexes containing Msh4 and Msh5 but does not require Spo16- and Spo22-dependent extension of synaptonemal complexes. In contrast, crossover assurance requires all ZMM proteins and full-length synaptonemal complexes.

During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. Rad52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the Rad52 independence of Dmcl, we reveal that Rac152 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of Rad52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that Rac152 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.

Meiotic recombination-related DNA synthesis (MRDS) was analyzed in Saccharomyces cerevisiae by specifically timed incorporation of thymidine analogs into chromosomes. Lengths and positions of incorporation tracts were determined relative to a known recombination hot spot along DNA, as was the timing and localization of incorporation relative to forming and formed synaptonemal complex in spread chromosomes. Distinct patterns could be specifically associated with the majority cross-over and non-cross-over recombination processes. The results obtained provide direct evidence for key aspects of current consensus recombination models, provide information regarding temporal and spatial relationships between non-cross-over formation and the synaptonemal complex, and raise the possibility that removal of RecA homolog Rad51 plays a key role in regulating onset of MRDS. Finally, classical observations on MRDS in Drosophila, mouse, and lily are readily mapped onto the findings presented here, providing further evidence for a broadly conserved meiotic recombination process.

The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, alpha and P, in the origins as specificity determinants and showed that when each component of the region A-site alpha pair and the region B-site beta pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site alpha. By using an electrophoretic mobility shift assay, we demonstrated that region B and site beta play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site alpha were involved in the efficient unwinding of several nucleotide residues around site ut, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.

Abstract The Mre11/Rad50/Xrs2 (MRX) complex is involved in DNA damage repair, DNA damage response, telomere control, and meiotic recombination. Here, we constructed and characterized novel mutant alleles of XRS2. The alleles with mutations in the C-terminal conserved domain of Xrs2 were grouped into the same class. Mutant Xrs2 in this class lacked Mre11 interaction ability. The second class, lacking a C-terminal end, showed defects only in telomere control. A previous study showed that this C-terminal end contains a Tel1-association domain. These results indicate that Xrs2 contains two functional domains, Mre11- and Tel1-binding domains. While the Mre11-binding domain is essential for Xrs2 function, the Tel1-binding domain may be essential only for Tel1 function in telomere maintenance. The third class, despite containing a large deletion in the N-terminal region, showed no defects in DNA damage repair. However, some mutants, which showed a reduced level of Xrs2 protein, were partially defective in formation of meiotic DSBs and telomere maintenance. These defects were suppressed by overexpression of the mutant Xrs2 protein. This result suggests that the total amount of Xrs2 protein is a critical determinant for the function of the MRX complex especially with regard to telomere maintenance and meiotic DSB formation.

Meiotic recombination requires the meiosis-specific RecA homolog Dmc1 as well as the mitotic RecA homolog Rad51. Here, we show that the two meiosis-specific proteins Mei5 and Sae3 are necessary for the assembly of Dmc1, but not for Rad51, on chromosomes including the association of Dmc1 with a recombination hot spot. Mei5, Sae3, and Dmc1 form a ternary and evolutionary conserved complex that requires Rad51 for recruitment to chromosomes. Mei5, Sae3, and Dmc1 are mutually dependent for their chromosome association, and their absence prevents the disassembly of Rad51 filaments. Our results suggest that Mei5 and Sae3 are loading factors for the Dmc1 recombinase and that the Dmc1-Mei5-Sae3 complex is integrated onto Rad51 ensembles and, together with Rad51, plays both catalytic and structural roles in interhomolog recombination during meiosis.

An E2 ubiquitin-conjugating enzyme, Rad6, working with an E3 ubiquitin ligase Bre1, catalyzes monoubiquitylation of histone H2B on a C-terminal lysine residue. The rad6 mutant of Saccharomyces cerevisiae shows a meiotic prophase arrest. Here, we analyzed meiotic defects of a rad6 null mutant of budding yeast. The rad6 mutant exhibits pleiotropic phenotypes during meiosis. RAD6 is required for efficient formation of double-strand breaks (DSBs) at meiotic recombination hotspots, which is catalyzed by Spo11. The mutation decreases overall frequencies of DSBs in a cell. The effect of the rad6 mutation is local along chromosomes; levels of DSBs at stronger hotspots are particularly reduced in the mutant. The absence of RAD6 has little effect on the formation of ectopic DSBs targeted by Spo11 fusion protein with a Ga14 DNA-binding domain. Furthermore, the disruption of the BRE1 as well as substitution of the ubiquitylation site of histone H2B also reduces some DSB formation similar to the rad6. These results suggest that Rad6-Bre1, through ubiquitylation of histone H2B, is necessary for efficient recruitment and/or stabilization of a DSB-forming machinery containing Spoil. Histone tail modifications might play a role in DSB formation during meiosis.

Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MATalpha recombines with the donor, MATalpha. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATalpha. We present evidence for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52.

RecA protein is involved in homology search and strand exchange processes during recombination. Mitotic cells in eukaryotes express one RecA, Rad51, which is essential for the repair of double-strand breaks (DSBs). Additionally, meiotic cells induce the second RecA, Dmc1. Both Rad51 and Dmc1 are necessary to generate a crossover between homologous chromosomes, which ensures the segregation of the chromosomes at meiotic division I. It is largely unknown how the two RecAs cooperate during meiotic recombination. In this review, recent advances on our knowledge about the roles of Rad51 and Dmc1 during meiosis are summarized and discussed. Copyright (C) 2004 S. Karger AG, Basel.

In Saccharomyces cerevisiae, the Rad52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways. We characterized a rad52 mutant, rad52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination. The rad52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats. We also analyzed the effect of the rad52-329 mutant on telomere recombination. Yeast cells lacking telomerase maintain telomere length by recombination. The rad52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination. In addition, we examined the roles of other recombination genes in the telomere recombination. The RAD51-independent recombination in the rad52-329 mutant is promoted by a paralogue of Rad52, Rad59. All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination. Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination. These findings indicate that Rad52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51.

We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1. These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51. Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 Mutant, but also the gamma-ray sensitivity of the mutant. Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type. The control of the cell cycle in response to meiotic DSBs is also discussed.

Two RecA-like rccombinases, Rad51 and Dmc1, function together during double-strand break (DSB)mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.

Two RecA homologs, Rad51 and Dmc1, assemble as cytologically visible complexes (foci) at the same sites on meiotic chromosomes. Time course analysis confirms that co-foci appear and disappear as the single predominant form. A large fraction of co-foci are eliminated in a red1 mutant, which is expected as a characteristic of the interhomolog-specific recombination pathway. Previous studies suggested that normal Dmc1 loading depends on Rad51. We show here that a mutation in TID1/RDH54, encoding a RAD54 homolog, reduces Rad51-Dmc1 colocalization relative to WT. A rad54, mutation, in contrast, has relatively little effect on RecA homolog foci except when strains also contain a tid1/rdh54 mutation the role of Tid1/Rdh54 in coordinating RecA homolog assembly may be very direct, because Tid1/Rdh54 is known to physically bind both Dmc1 and Rad51. Also, Dmc1 foci appear early in a tid1/rdh54 mutant. Thus, Tid1 may normally act with Rad51 to promote ordered RecA homolog assembly by blocking Dmc1 until Rad51 is present. Finally, whereas double-staining foci predominate in WT nuclei, a subset of nuclei with expanded chromatin exhibit individual Rad51 and Dmc1 foci side-by-side, suggesting that a Rad51 homo-oligomer and a Dmc1 homo-oligomer assemble next tb one another at the site of a single double-strand break (DSB) recombination intermediate.

Background: DMC1, the meiosis-specific eukaryotic homologue of bacterial recA, is required for completion of meiotic recombination and cell cycle progression past prophase. In a dmc1 mutant, double strand break recombination intermediates accumulate and cells arrest in prophase. We isolated genes which, when present at high copy numbers, suppress the meiotic arrest phenotype conferred by dmc1 mutations. Results: Among the genes isolated were two which suppress arrest by altering the recombination process. REC114 suppresses formation of double strand break (DSB) recombination intermediates. The low viability of spores produced by dmc1 mutants carrying high copy numbers of REC114 is rescued when reductional segregation is bypassed by mutation of spo13. High copy numbers of RAD54 suppress dmc1 arrest, promote DSB repair, and allow formation of viable spores following reductional segregation. Analysis of the combined effects of a null mutation in RED1, a gene required for meiotic chromosome structure, with null mutations in RAD54 and DMC1 shows that RAD54, while not normally important for repair of DSBs during meiosis, is required for efficient repair of breaks by the intersister recombination pathway that operates in red1 dmc1 double mutants. Conclusions: Over-expression of REC114 suppresses meiotic arrest by preventing formation of DSBs. High copy numbers of RAD54 activate a DMC1-independent mechanism that promotes repair of DSBs by homology-mediated recombination. The ability of RAD54 to promote DMC1-independent recombination is proposed to involve suppression of a constraint that normally promotes recombination between homologous chromatids rather than sisters.

Background: The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double-strand break (DSB) repair and meiotic recombination. A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange. Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions. Very little has been discovered about the biochemical roles and structure of the Rad52 protein. Results: Purified Rad52 protein binds to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Electron microscope observations revealed that Rad52 molecules form multimeric rings. An increase in the intensity of fluorescence when Rad52 is bound to epsilon DNA showed an alteration of the structure of ssDNA. RPA was binding to Rad52 and enhanced the annealing of complementary ssDNA molecules. This enhancement was not observed in Escherichia coli SSB protein of T4 phage gp32 protein. Conclusion: Rad52 forms a ring-like structure and binds to ssDNA. Its structure and DNA binding properties are different from those of Rad51. The interaction of Rad52 with RPA plays an important role in the enhancement of annealing of complementary ssDNAs. We therefore propose that Rad52 mediates the RAD51-independent recombination through an ssDNA annealing, assisted by RPA.

The RAD54 gene, which encodes a protein in the SW12/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae. The yeast genome project revealed a homologue of RAD54, RDD54/TLD1. Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis. The rad54 mutant is sensitive to the aIkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion. The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis. However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant. These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of RAD54. In meiosis, both single mutants produce viable spores at slightly reduced frequency. However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants. Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type. These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins.

The ColE2-P9 Rep protein specifically binds to the origin and initiates DNA synthesis. Interaction of the Rep proteins with the origins of plasmids ColE2-P9 and ColE3-CA38 (one of the close relatives of ColE2-P9) is plasmid-specific. By using chimeric rep genes and chimeric origins we showed that the two region, A and B, in the C-terminal regions of the Rep proteins and the two sites, alpha and beta, in the origins are important for the determination of specificity. When each of the A/alpha and B/beta Fairs is from the same plasmids, the plasmid replication is efficient. On the other hand, if only the A/alpha pair is from the same plasmids, the plasmid replication is inefficient. For the region A, the plasmid-specificity is mainly determined by the presence or absence of a nine-amino acid sequence. For the region B, the specificity is probably determined by several amino acids. The region B contains a segment of amino acid sequence which shows significant homology with the DNA recognition helices of various DNA binding proteins. At the site alpha, the single additional base-pair in the ColE3-CA38 origin can be either A/T or T/A. At the site beta, however, the single additional base-pair in the ColE2-P9 origin must be G/C. Among other possibilities we propose that the region A is a linker connecting the two domains in the Rep protein involved in DNA-binding and that the region B is apart of the sequence-specific DNA-binding domain. (C) 1996 Academic Press Limited

出芽酵母は、複製起点、セントロメア、テロメアの染色体機能の最小領域をもちいて完全に人工合成の染色体（人工ミニ染色体）を構築できる。人工ミニ染色体は減数第一分裂期で高頻度の不分離を示す一方で第二分裂では正常に分配することを示した。また、不分離の原因は減数分裂期交叉型組換え欠損によると考えられたため人工ミニ染色体上にGAL1プロモーターのUASを挿入しGal4BD-Spo11発現株において解析を行ったが改善はみられなかった。一方で、染色体軸因子のMek1キナーゼの自己リン酸化による活性化は減数分裂期組換えの鋳型選択に重要な機能を果たしその変異株では異所性の組換えが高頻度で起きることを明らかにした。

染色体が調和して機能する仕組みを染色体オーケストレーションシステム（染色体OS） と呼び、精子、卵子などの配偶子形成に必須な減数分裂では染色体OSがより劇的に変化する。減数分裂期には染色体3D構造が大きく変化するだけでなく、染色体を場として組換えなどの様々なDNA代謝反応が“制御された形”で起き、その制御プラットフォームが染色体OSであると考えられる。本計画研究は減数分裂期染色体を染色体OSのモデル系として捉え、減数分裂期染色体構造、特にその基盤となる染色体軸―ループ構造と機能を理解し、減数分裂期特異的染色体上で起きる減数分裂期組換えの制御の分子メニズムを明らかにすることを目的とする。2018年度では、染色体構造とその上で起こる組換えの連携の分子メカニズムを解明することを目標とし、超解像度蛍光顕微鏡を使用して、減数分裂期の染色体構造、特に高次構造（軸―ループ構造）を、減数分裂期染色体の軸構成要素であるコヒーシンの局在を解析した結果、減数第一、第二分裂期に起こる切断とは異なる、切断に依存しない仕組みで、コヒーシン複合体が染色体から減数分裂第１分裂後期で解離すること、その解離には特異的なリン酸化酵素Polo-likeキナーゼ(PLK)が必要であることに加えて、Dbf4依存性Cdc7キナーゼ(DDK)が必要であり、この２つのキナーゼの協調的は働きにより、コヒーシン制御因子の１つWAPL(Rad61)とRec8がリン酸化されることが、この解離に必要であることを見出した。また、この切断非依存性のコヒーシンの解離に伴い、染色体がコンパクトに凝集する構造変換が起こることも分かった。この染色体構造変換は、染色体分配に必須のキアズマ（相同組換え）形成と関連すると考えられる。減数分裂期の染色体構造形成に関しての新しいメカニズムを提示できる可能性が高い。

M期では他の細胞周期と異なり、DSB（DNA二重鎖切断）修復が起こると大きなゲノムの不安定化をひきおこし、これを積極的に抑制する機構があることがわかってきた。申請者は非相同末端結合因子XRCC4のリン酸化を介したDSB修復抑制機構を明らかにしてきた。さらにXRCC4を破壊すると細胞分裂に影響する予備データを得ていた。本研究ではさらに分子レベルでの機構解明へと研究を深め、XRCC4のリン酸化の分子機構を明らかにすること、またこの時期わずかに起こるDSB修復の塩基レベルでの解析を行いこの修復の意義を理解すること、さらにDSB修復因子と細胞分裂との関わりを明らかにし、この時期の特殊な修復機構と連携した正確に染色体を分配する仕組みによる新規ゲノム安定性維持機構を明らかにすることを目的とした。本年度、XRCC4のリン酸化の分子機構を明らかにするために生化学的な解析のセットアップ、M期に起こるDSB修復の検出系を作製したが退職のため発表に十分なデータを得ることができなかった。

DNA二重鎖切断(DSB)はDNAの両鎖の遺伝情報が失われることから最も重篤なDNA傷害のひとつである。また、ヒトでDSB修復に関わる蛋白質(Nbs1, Mre11, LigaseIV)が機能しなくなると高発がん性遺伝病となることから、DSBが染色体不安定化の引き金となると考えられる。DSBは組換え修復によって修復されるが、ヒト細胞内における組換え過程を理解する上で、障壁となっているのが、高等真核生物においてDSB修復の素過程の分子メカニズムを解析するための優れた系が存在しないことである。そこで、我々はその問題を解決するために新規のDSB導入・検出法を開発しそれを用いてヒトでの組換え修復の素過程の理解にむけた研究を行う。 今年度は、「制限酵素を用いたDNA切断導入法の確立」を主たる目的として、制限酵素の細胞内への導入方法について条件検討を行った。また、人工的に導入されるDSBの分布をハイスループットDNAシーケンスによって同定するための効率の良いDSB末端の回収方法について条件検討を行った。その結果、全ての染色体由来のDSB断片を効率よく回収することに成功した。今後は回収したDSB断片の塩基配列を決定し、ゲノム上にマッピングを行うことでDSBの導入される頻度を明らかにする。また、制限酵素によって導入されたDSB末端における、既知の修復タンパク質(γH2AX、Rad51など)の会合状態について調べるために間接蛍光抗体法の条件について改良を行った。その結果、界面活性剤と固定剤で細胞を処理することでクロマチン上の修復タンパク質の検出感度を上げることができた。

染色体安定維持機構において最も重要なポイントは単鎖DNA形成のコントロールである。DNA二重鎖切断(DSB)修復経路はNHEJと相同組換え(HR)の二つが主たる経路であるが、エラーフリーの修復系であるHRではDSB末端の単鎖化というリスクが伴う。そのリスクを以下に最小にするかが染色体安定維持機構における重要点である。我々はこれまでHRを主な修復系とするS/G2期において、NHEJ因子がDSB末端において単鎖DNA形成を抑制しており、その制御にCDK1の活性が重要な機能を果たすことを明らかにしてきた。 我々の知見はG1期の修復においてのみ重要とされて来たNHEJのS/G2期での新しい機能の可能性とDNA傷害修復の新しい制御メカニズムを提唱するものである。ここでは、NHEJ因子がどのようにDSB末端の単鎖化を制御しているのか明らかにする。 1.NHEJ因子LiflのCDKによるリン酸化の機能の解明 今までの解析から、NHEJに必須のDNAリガーゼIV複合体のサブユニットのLiflの変異株ではHRの開始反応であるDSBの単鎖化の開始が遅れることを明らかにした。単鎖化の開始はSae2タンパク質が、担っていることから、Liflタンパク質とSae2タンパク質の相互作用について解析を行ったところ、両者は物理的に相互作用することを明らかにした。そこで、その相互作用部位を特定したところC末端領域の種間で保存されている領域が必要であった。しかし、単鎖化の活性に必要なドメインとは重なっていなかったことから、相互作用に欠損を示すsae2変異株を作成し、DSB末端の単鎖化のスピードについて解析を行ったところ野生株と比較して単鎖化が遅れることを明らかにした。つまりNHEJ因子とHRの開始因子であるSae2がDSB末端で相互作用することが効率の良いDSBの単鎖化に必要であることを示唆している。

減数分裂期の大きな特徴のひとつは、染色体の構造が大きく変化し,また、ダイナミックな運動を伴うことである。中でもシナプトネマ複合体(synaptonemal complex;以下SC)は、減数分裂期特有の染色体構造であり,酵母からヒトまで広く保存された染色体構造である体細胞期に必須の役割を果たすSCFの減数分裂期における機能を知るために、減数分裂期特異的にSCFのサブユニット(Cdc53)の発現を抑制する株を作成して解析を行った。その結果、シナプトネマ複合体形成に欠損がみられ、特に軸構造の構成因子Rec8の局在に異常が見られた。Rec8は減数分裂期特異的なコヒーシンのサブユニットであり、その体細胞分裂期のカウンターパートはScclである。減数分裂期に入るとScclからRec8に置き換わるが、そのしくみはわかっていない。Rec8の局在が異常であったことから、Scclが減数分裂期染色体に残存している可能性について検討したがScclの局在は観察されなかった。この結果から、SCF変異株では姉妹染色体接着が完全ではない可能性が示唆された。 一方、SCFによるユビキチン化の多くはサイクリン依存的キナーゼ(CDK)に依存して起こることが知られている。そこで、CDK活性がシナプトネマ複合体形成に必要かどうか、ATPアナログ高感受性CDC28変異株、cdc28-aslを用いて解析を行った。その結果、CDKによるリン酸化はシナプトネマ複合体形成に必須であることが明らかとなった。これらの結果から、CDKによるリン酸化とそれに依存したSCFによるタンパク質のユビキチン化がシナプトネマ複合体形成の特に軸構造の形成過程に重要な機能を担っていると考えられる。

生殖細胞形成の大きな目的は次世代へのゲノム情報を伝える1倍体の配偶子を作ることである。配偶子は減数分裂を経て形成されるが、中でも減数第一分裂では組換えが高頻度で起き、染色体もシナブトネマ複合体(SC)を形成することで、相同染色体の分配を促進している。近年SCが出来るパキテン期が減数分裂期の細胞周期の進行に大切な役割を果たすことが注目されている。特にパキテン期は染色体形態形成、組換えの状態をモニターし、減数分裂期の進行を保証するチェックポイントがある。しかし、その実体についてはほとんど分かっていない。さらに興味深いのがこのcheckpointにヒストンH3 K79(ほ乳類ではK76)のメチル化酵素Dotlが関わることである。また、Dotlは減数分裂期組換えの開始であるDNA2重鎖切断の形成にも関わることが知られている。本研究ではSetl,Dotlの新しい機能として減数分裂期特異的染色体構造であるシナプトネマ複合体に関わること,特に染色体の軸構造形成に重要であることを明らかにした。シナプトネマ複合体にヒストンの修飾、あるいはこれらメチル基転移酵素による別のタンパク質の修飾が関与すると考えている。さらに、Dotl,Setlの上流で働くと考えられヒストンH2BのK4のユビキチンに関わると考えられるPafl複合体もDNA2重鎖切断の形成やシナプトネマ複合体に関わることを明らかにできた。Pafl複合体は転写の伸長にも関わり、転写と減数分裂期の染色体反応の共役と言う点で興味深い。

DNA二重鎖切断(DSB)は両鎖の遺伝情報を同時に失うことから最も重篤なDNA損傷だと考えられている。その傷を修復する手段は主に2つある。一つは、相同組換え(HR)でもう一つは、非相同末端結合(NHEJ)である。相同組換えは同じ遺伝情報をもつ姉妹染色体をコピーする反応であることからエラーフリーの修復システムであるが、損傷末端をエキソヌクレアーゼによって、1Kbにもわたって単鎖化する必要がある。一方のNHEJは末端をDNAリガーゼによって再結合させろ単純な修復糸であるか末端部分の遺伝情報が一部失われる危険がある。今年度の解析によって、HRを主な修復系とするS/G2期において、NHEJ因子Lif1タソパク質がDSB末端において単鎖DNA形成を制御しており、その制御にCDK1の活性が重要な機能を果たすことを出芽酵母の部位特異的DSBの系を用いて明らかにした。また、LDKによるリン酸化を受けないLif1変異株においてはDSB末端の単鎖化を伴うNHEJ活性が特異的に低下していることを明らかにした。これらの我々の知見はG1期の修復においてのみ重要とされて来たNHEJのS/G2期での新しい機能の可能性とDNA傷害修復の新しい制御メカニズムを提唱するものである。HRが主たる修復経路となるS/G2期で、末端単鎖化を伴う形でのマイクロホモロジーを用いたNHEJがHRのバックアップジステムとして存在する可能性を示唆しており、その制御にCDK1によるリン酸化がかかわり細胞周期による制御を可能にしていると考えられる。

本研究の目標はヒト細胞内におけるDNA二重鎖切断の修復の過程を明らかにする上で、今必要なものは、ゲノム上に部位特異的に同調的にDNA二重鎖切断(DSB)を導入しその修復過程の経時変化をモニターすることができる系を確立することである。ヒト培養細胞HeLaS3細胞およびヒト正常リンパ球細胞を用いて、制限酵素BamHIにより生細胞中でDSBを導入させ、その修復過程について解析を行った。今年度は、おもに制限酵素により導入されたDSB部位のヒトゲノム上での位置の特定を行うことを目標として解析を行った。DSB末端に相補的な末端を持つ、ビオチンラベルしたオリゴDNAによりキャッピングを行い、ストレプトアビジンビーズにより回収してDSB末端の塩基配列を決定した。その結果、ヒトゲノム上で約200カ所の高頻度でDSBが入る部位を特定することができた。その中でも特に頻度が高かった部位について、クロマチン免疫沈降法を行った。DSB末端プールからDSB依存的に特定領域を検出することができたが、修復タンパク質に対する抗体を用いた、免疫沈降産物からのクロマチン免疫沈降法特定領域の検出にはいたらなかった。さらに検出条件の検討を行うことにより、改善可能だと考えている。逆に特定部位にこだわらず、全ゲノム上のDSB末端の濃縮方法を確立したことからゲノムワイドなDSBの分布とゲノム上の構造あるいは様々な染色体上の蛋白質の分布との比較が可能になった。ゲノム安定化における脆弱部位の特定や、今後、この手法を用いてゲノム安定化に寄与するタンパク質とその修飾状態等の要因究明に多いに貢献できると考えている。

減数分裂期に起こるゲノムの変化は相同組換えにより生じる。組換えは親の持つ遺伝情報を混ぜ合せることで多様性を生み出すばかりでなく,減数第1分裂における相同染色体の分配に必須であることも知れられている。減数分裂期の相同組換えは、体細胞分裂期の組換えと異なり,複雑な制御を受けていると言った特徴がある。本研究では、減数分裂期のおける組換えに関わるいくつかの因子を同定することで、そのメカニズムの解明を行った。特に、染色体運動と構造が組換えを制御しているという仕組みを明らかにした。

減数分裂期に発現するSpo16 タンパク質が、シナプトネマ複合体と呼ばれる減数分裂期に形成される染色体高次構造体の新規構成因子であることを明らかにした。配偶子を作るとき、染色体数を半数にするために、キアズマを染色体上で正しく配置する仕組み(干渉)と全ての染色体にキアズマを必ず作る仕組み(保証)がある。シナプトネマ複合体が干渉と保証の両方を制御するがそれぞれに異なるユニットを用いることを明らかにした。

減数分裂期には相同染色体間で交叉型組換え(Crossover)を行うことが正常に還元分裂を行うために必須のイベントである。そのためにCrossoverは通常の組換え反応にはない厳密な制御下で行われている。そこで、このCrossover形成とその制御がどのように行われているかについて解析を行った。本研究によって酵母ATRホモログMec1がCrossover形成制御に関わっていることを明らかにした。また、その制御にはMec1のKinase活性が必須であることをMEC1のKinase dead変異株の解析から明らかにした。また、以前の解析からCrossoverの効率的な形成とその制御に2つのRecAホモログRad51とDmc1の協調的な機能が必要であることを示したが、ATPase活性を欠いたRad51変異株の解析から減数分裂期組換えにはRad51のATPase活性は必要ないことを明らかにした。Rad51はDmc1のリクルートに必要であることから、Rad51は減数分裂期組換えにおいては組換えの初期反応とDmc1のリクルートには必要だが、ATPase活性が必要となるDNA鎖交換反応は主にDmc1が行っていることを示唆している。体細胞分裂期の組換えではRad51がDNA鎖交換反応を行うことからこの違いが体細胞分裂期と減数分裂期の組換えの質の違いを生んでいると考えられる。これらの結果から、減数分裂期にはRad51 ATPase活性を抑制するメカニズムが存在すると考えられ、Mec1が直接的か間接的にRad51の活性をコントロールしている可能性が示唆された。

染色分体早期解離(PCS)症候群は我が国で発見された染色体不安定症候群で、患児細胞は染色分体早期解離と多彩な異数体モザイクを示す。これまでに日本人9家系13症例が報告され、日本人患児13例全例が重度小頭症と発育遅滞、小脳虫部の低形成を伴うDandy-Walker奇形などを示し、全例が生後数ヶ月から難治性けいれんを発症した。本研究の目的は、PCS患者細胞におけるM期紡錘体チェックポイント異常の分子機構を解明し、細胞周期制御の破綻による腫瘍化のメカニズムを明らかにすることである。以下の諸点を明らかにした。 1)3例のPCS患者から皮膚線維芽細胞を採取して不死化細胞株を樹立した。 2)種々のM期チェックポイント蛋白およびセントロメア蛋白の細胞内局在を免疫染色法で解析して、BubR1とp55cdcのキネトコアシグナルがほぼ消失していることを明らかにした。 3)PCS細胞株にBUB1B遺伝子が存在する15番染色体を移入すると、BubR1とp55cdcのキネトコアへの集積および紡錘体チェックポイントが正常化した。 4)PCS患者サンプルでBUB1B遺伝子解析を行った。7家系中4家系に同一の一塩基欠失を、3家系にそれぞれスプライス変異、ナンセンス変異、ミスセンス変異を同定した。一方、同定された7家系の変異はすべてヘテロ接合であり、反対側のアレルに変異は検出されなかった。 5)7家系の変異が検出されないBUB1Bアレルはほぼ共通したハプロタイプを示し、BubR1タンパク発現量が低下していることを見いだした。以上の所見から、PCS症候群はBubR1発現量の50%以上の低下が原因であることが明らかとなった。

ナイミーヘン症候群(NBS)は高発がん性や電離放射線高感受性そして染色体不安定性を特徴とするヒト劣性遺伝病である。原因遺伝子NBS1がヌクレアーゼ活性もDNA結合領域も有しない修復蛋白をコードしている事を我々は既に報告した。本研究では、NBS1蛋白C末側の真核生物で保存されている領域で相同組換えに重要なMre11/Rad50ヌクレアーゼ複合体と結合していることを示した。また、我々はヒストンH2AバリアントのH2AXが放射線照射後直ちにリン酸化を受けることに注目して、細胞内でH2AXとNBS1蛋白が結合していることをin vitro実験や細胞を用いた実験で示した。特にNBS1蛋白N末側のFHA/BRCTドメインとピストンが直接結合していることを確認した。この結果、FBS1はMre11/Rad50複合体をリン酸化ヒストンを目印として放射線誘発のDNA二重鎖断部位にリクルートして相同組換えを開始するモデルが提示された。実際、NBS患者細胞およびNBS1ノックアウト動物細胞では相同組換え能が顕著に低下していることがSCneoなどのレポーター遺伝子を用いた解析から明らかになった。また、NBS1蛋白のN末およひC末側の欠失は、コヒーシンSMC1との結合ならびにS期チェックポイントに必要なSMC1リン酸化の阻害をもたらした。さらにNBS1はBRCA1やワーナー症候群の蛋白WRNとも結合して損傷部位で巨大複合体を形成する事、そしてこの複合体形成の阻害が染色体不安定性をもたらす事が判明した。今後、この複合体形成が修復Fidelityやチェックポイントとのロストークに果たす役割およびその発がんにおける意義について解析する。

染色分体早期解誰症候群(PCS syndrome)は我が国で発見された染色体不安定症候群で、ヒトにおける初めてのM期紡錘体形成チェックポイント欠損症である。患者由来の細胞は、染色体分析で姉妹染色分体が高頻度に解離したpremature chromatid separation(PCS)と多彩な異数性モザイクを示す。我々はPCS症候群の紡錘体形成チェックポイント異常のメカニズムを解明することを目的に、種々のキネトコア蛋白および紡錘体チェックポイント蛋白の発現と細胞内局在をウエスタンブロット法および免疫染色法で詳細に解析した。これまでに以下の諸点を明らかにした。 1.3例のPCS症候群患者の不死化細胞株を樹立した。患者細胞はいずれもBubR1およびp55cdc蛋白のキネトコアへの集積がほぼ消失していることを見いだした。 2.BubR1遺伝子の存在するヒト15番染色体をPCS患者細胞へ導入すると、BubR1・p55cdcのキネトコアシグナルが正常化するとともに、M期紡錘体形成チェックポイントも正常化することが確認された。 3.PCS患者細胞ではBubR1に遺伝子変異は認めないが、BubR1からp55cdcへのM期紡錘体チェックポイントの最終経路に機能異常があり、この経路に関わる未知の因子が欠損している可能性が考えられた。 本年度の研究により、PCS症候群はBubR1からp55cdcへのM期紡錘体チェックポイントの最終経路に機能異常があることが明らかとなった。この経路に関わる未知の因子が欠損している可能性が強く示唆された。

ゲノムDNAの多様化に関与する遺伝的組換えの機能は、ゲノムDNAの切断傷害の修復、DNA複製阻害の除去や染色体のテロメア構造の維持にも必須である。組換えの中心蛋白質、Rad51、Rad52やMre11/Rad50/Xrs2複合体(MRX複合体)が、どのような仕組みでそれぞれの現象に関与しているかを明らかにして、遺伝情報の安定な維持と多様化の仕組みの総合的な理解を目指した。以下は最も代表的なものに限定した成果である。 1.Mre11複合体の機能制御を行っているXrs2機能ドメインの詳細な解析を行い、(1)Xrs2はそのC端に近い、32アミノ酸ドメイン(MBX)でMre11に結合し、核内にMre11を移行させる。(2)DNA傷害の修復には、Mre11が核内にあればXrs2は必要ない。(3)テロメア複製と減数分裂期組換えには、MBXの他に、それに隣接するC端側104、及びN端側49のアミノ酸領域がそれぞれ必要である。2.DNA二重鎖切断に特異的なTell-Mre11チェックポイント経路を発見した。体細胞分裂期にはRad53、Rad9が、減数分裂期にはMre4/Mek1がこの経路に働く。Mre11複合体は切断を感知し経路を誘導し、またこの経路で活性制御を受けながら組換えを進行させる。組換え開始DNA二重鎖切断の前に先ずMre11/Xrs2が染色体にリクルートされ、その後Rad50がそこへリクルートされる。3.DNAの相同性の検索にはRad51-Rad52-DNA単鎖複合体が必要であること、Rad52が組換え反応の最終段階で最終産物の生成に必要であることが分かった。4.減数分裂稀組換えに特異的に関与するヘリカーゼ遺伝子MER3を発見した。この欠損株は交差型組換え体の頻度だけを低下させるので、組換え体の交差型か遺伝子変換型下が決まるのは、組換え中間体形成途上であることが強く示唆された。

Nbs1遺伝子欠損および遺伝子二重欠損細胞の作成:Nbs1遺伝子欠損ヘテロマウスをAtmまたはKn70遺伝子ヘテロ欠損マウスとそれぞれ交配してF2マウスを誕生させ、尻尾からDNAを抽出してPCR法で二重ヘテロ欠損マウスを同定した。得られた二重ヘテロ欠損マウスを交配して8.5日齢マウス胚を採取し、Nbs1/AtmおよびNbs1/Ku70の遺伝子二重欠損細胞株同定を進めている。 遺伝子欠損細胞の機能解析:Ku70欠損細胞と、Atm欠損細胞とNbs1欠損細胞について、以下の表現型の差異を比較検討した。 (i)放射線誘発染色体構造異常:細胞に2Gyのガンマ線を照射して24時間培養後、コルセミド処理して染色体標本を作成する。100細胞の染色体核板を観察し染色分体異常の出現頻度を計測した。 (ii)放射線致死感受性:細胞に1、2、4Gyのガンマ線を照射してディシュに播種・培養し、約2週間後に出現するコロニー数を計測して生存率曲線を作成した。いずれの細胞株も正常細胞に比べ、放射線高感受性であることを確認した。