Homologs of MutS and MutL during mammalian meiosis

In eukaryotes, homologs of the Escherichia coli MutS and MutL proteins are crucial for both meiotic recombination and post-replicative DNA mismatch repair. Both pathways require the formation of a MutS homolog complex which interacts with a second heterodimer, composed of two MutL homologs. During mammalian meiosis, it is likely that chromosome synapsis requires the presence of a MSH4-MSH5 heterodimer. PMS2, a MutL homolog, seems to play an important role in this process. A MSH4-MSH5 heterodimer is also likely present later with other MutL homologs (MLH1 and MLH3) and is involved in the crossing-over process. The phenotype of msh4-/- mutant mice and MSH4 immunolocalization on meiotic chromosomes suggest that MSH4 has an early function in mammalian meiotic recombination. Both MSH4 and PMS2 directly interact with the RAD51 DNA strand exchange protein. In addition, MSH4 and RAD51 proteins co-localize on mouse meiotic chromosome cores. These results suggest that MSH4 and its partners could act, just after strand exchange promoted by RAD51, to check the homology of DNA heteroduplexes.     

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.     

ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.     

MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp

In the yeast Saccharomyces cerevisiae, mismatch repair (MMR) is initiated by the binding of heterodimeric MutS homolog (MSH) complexes to mismatches that include single nucleotide and loop insertion/deletion mispairs. In in vitro experiments, the mismatch binding specificity of the MSH2-MSH6 heterodimer is eliminated if ATP is present. However, addition of the MutL homolog complex MLH1-PMS1 to binding reactions containing MSH2-MSH6, ATP, and mismatched substrate results in the formation of a stable ternary complex. The stability of this complex suggests that it represents an intermediate in MMR that is subsequently acted upon by other MMR factors. In support of this idea, we found that the replication processivity factor proliferating cell nuclear antigen (PCNA), which plays a critical role in MMR at step(s) prior to DNA resynthesis, disrupted preformed ternary complexes. These observations, in conjunction with experiments performed with streptavidin end-blocked mismatch substrates, suggested that PCNA interacts with an MSH-MLH complex formed on DNA mispairs.     

CRM1-dependent nuclear export and dimerization with hMSH5 contribute to the regulation of hMSH4 subcellular localization

MSH4 and MSH5 are members of the MutS homolog family, a conserved group of proteins involved in DNA mismatch correction and homologous recombination. Although several studies have provided compelling evidences suggesting that MSH4 and MSH5 could act together in early and late stages of meiotic recombination, their precise roles are poorly understood and recent findings suggest that the human MSH4 protein may also exert a cytoplasmic function. Here we show that MSH4 is present in the cytoplasm and the nucleus of both testicular cells and transfected somatic cells. Confocal studies on transfected cells provide the first evidence that the subcellular localization of MSH4 is regulated, at least in part, by an active nuclear export pathway dependent on the exportin CRM1. We used deletion mapping and mutagenesis to define two functional nuclear export sequences within the C-terminal part of hMSH4 that mediate nuclear export through the CRM1 pathway. Our results suggest that CRM1 is also involved in MSH5 nuclear export. In addition, we demonstrate that dimerization of MSH4 and MSH5 facilitates their nuclear localization suggesting that dimerization may regulate the intracellular trafficking of these proteins. Our findings suggest that nucleocytoplasmic traffic may constitute a regulatory mechanism for MSH4 and MSH5 functions.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.     

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.     

hMSH4-hMSH5 recognizes Holliday Junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes

Five MutS homologs (MSH), which form three heterodimeric protein complexes, have been identified in eukaryotes. While the human hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers operate primarily in mitotic mismatch repair (MMR), the biochemical function(s) of the meiosis-specific hMSH4-hMSH5 heterodimer is unknown. Here, we demonstrate that purified hMSH4-hMSH5 binds uniquely to Holliday Junctions. Holliday Junctions stimulate the hMSH4-hMSH5 ATP hydrolysis (ATPase) activity, which is controlled by Holliday Junction-provoked ADP-->ATP exchange. ATP binding by hMSH4-hMSH5 induces the formation of a hydrolysis-independent sliding clamp that dissociates from the Holliday Junction crossover region, embracing two homologous duplex DNA arms. Fundamental differences between hMSH2-hMSH6 and hMSH4-hMSH5 Holliday Junction recognition are detailed. Our results support the attractive possibility that hMSH4-hMSH5 stabilizes and preserves a meiotic bimolecular double-strand break repair (DSBR) intermediate.     

金属离子促进嗜热四膜虫错配修复蛋白Mlh3核酸内切酶活性

嗜热四膜虫有性生殖过程中生殖系小核延伸并活跃转录,减数分裂过程中染色体同源重组起始于程序化的DNA双链断裂的形成,DNA错配修复系统能够去除DNA复制过程中所引起的错配并促进同源重组。减数分裂特异表达的错配修复因子Mlh3对四膜虫的有性生殖是必需的,然而具体功能并不清楚。本研究人工合成MLH3（TTHERM_001044369）基因,构建重组表达质粒pGEX-MLH3, 转化大肠杆菌BL21（DE3）并获得重组表达的GST-Mlh3蛋白。纯化的GST-Mlh3蛋白在配位不同的金属离子Cu2+、Mn2+、Mg2+后,有效切割超螺旋质粒DNA。ATP和ADP可进一步促进Mlh3的核酸内切酶活性。突变Mlh3中离子结合模体DQHA(X)2E(E)4E中的D117和E123位点,Mlh3D117N/E123A的核酸内切酶活性降低。进一步删除离子结合和ATP结合位点的C端结构域,突变体的核酸内切酶活性进一步降低,表明Mlh3的核酸内切酶活性是离子依赖型。减数分裂期HA-Mlh3免疫共沉淀鉴定了Mlh3可能的相互作用因子链交换蛋白Dmc1、DSB修复蛋白Mnd1、MutS、染色体维持蛋白Smc2和Smc4。结果表明,四膜虫的Mlh3通过金属离子依赖的内切酶活性,以及与其他因子相互作用,在减数分裂错配修复和同源重组过程中发挥重要作用。     

Single-molecule views of MutS on mismatched DNA

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.     

人精母细胞重组频率和年龄相关性的分析

减数分裂遗传重组对同源染色体的正确分离和单倍体的正确形成起至关重要的作用,但人们对人精母细胞减数分裂遗传重组机制了解的还很少。通过免疫荧光染色技术标记减数分裂I联会复合体上的MLH1(DNA错配修复蛋白)位点可以检测人精母细胞的重组。文章对10例可育男性进行分析,发现每个细胞中重组位点数平均为49.4士4.4,范围为33~63,具有显著的个体差异,只有0.4%(1/220)的常染色体SC上缺少MLH1位点。进一步通过Spearman相关性分析,分析了年龄因素与个体间重组位点差异的相关性,结果提示年龄因素对常染色体及性染色体的重组均无影响。     

Formation of hMSH4-hMSH5 heterocomplex is a prerequisite for subsequent GPS2 recruitment

Increasing evidence suggests that components of the DNA mismatch repair (MMR) pathway play multifunctional roles beyond the scope of mismatch correction, including the modulation of cellular responses to DNA damage and homologous recombination. The heterocomplex consisting of MutS homologous proteins, hMSH4 and hMSH5, is believed to play essential roles in meiotic DNA repair particularly during the process of meiotic homologous recombination (HR). In order to gain a better understanding of the mechanistic basis underlying the roles of these two human MutS proteins, we have identified G-protein pathway suppressor 2 (GPS2) (i.e., an integral component of a deacetylase complex) as an interacting protein partner specifically for the hMSH4-hMSH5 heterocomplex. The interaction with GPS2 is entirely dependent on the physical association between hMSH4 and hMSH5, as disruption of the interaction between hMSH4 and hMSH5 completely abolishes GPS2 recruitment. Our analysis further indicates that the association with GPS2 is mediated through the interface of hMSH4-hMSH5 complex and the N-terminal region of GPS2. Moreover, these three proteins interact in human cells, and analysis of microarray data suggested a coordinated expression pattern of these genes during the onset of meiosis. Together, the results of our present study suggest that the GPS2-associated deacetylase complex might function in concert with hMSH4-hMSH5 during the process of homologous recombination.     

Mouse HFM1/Mer3 Is Required for Crossover Formation and Complete Synapsis of Homologous Chromosomes during Meiosis

Faithful chromosome segregation during meiosis requires that homologous chromosomes associate and recombine. Chiasmata, the cytological manifestation of recombination, provide the physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Formation of most crossover (CO) events requires the assistance of a group of proteins collectively known as ZMM. HFM1/Mer3 is in this group of proteins and is required for normal progression of homologous recombination and proper synapsis between homologous chromosomes in a number of model organisms. Our work is the first study in mammals showing the in vivo function of mouse HFM1. Cytological observations suggest that initial steps of recombination are largely normal in a majority of Hfm1^−/− spermatocytes. Intermediate and late stages of recombination appear aberrant, as chromosomal localization of MSH4 is altered and formation of MLH1foci is drastically reduced. In agreement, chiasma formation is reduced, and cells arrest with subsequent apoptosis at diakinesis. Our results indicate that deletion of Hfm1 leads to the elimination of a major fraction but not all COs. Formation of chromosome axial elements and homologous pairing is apparently normal, and Hfm1^−/− spermatocytes progress to the end of prophase I without apparent developmental delay or apoptosis. However, synapsis is altered with components of the central region of the synaptonemal complex frequently failing to extend the full length of the chromosome axes. We propose that initial steps of recombination are sufficient to support homology recognition, pairing, and initial chromosome synapsis and that HFM1 is required to form normal numbers of COs and to complete synapsis.     

Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements

Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.     

The Arabidopsis MutS homolog AtMSH5 is required for normal meiosis

MSH5, a member of the MutS homolog DNA mismatch repair protein family, has been shown to be required for proper homologous chromosome recombination in diverse organisms such as mouse, budding yeast and Caenorhabditis elegans. In this paper, we show that a mutant Arabidopsis plant carrying the putative disrupted AtMSH5 gene exhibits defects during meiotic division, producing a proportion of nonviable pollen grains and abnormal embryo sacs, and thereby leading to a decrease in fertility. AtMSH5 expression is confined to meiotic floral buds, which is consistent with a possible role during meiosis. Cytological analysis of male meiosis revealed the presence of numerous univalents from diplotene to metaphase I, which were associated with a great reduction in chiasma frequencies. The average number of residual chiasmata in the mutant is reduced to 2.54 per meiocyte, which accounts for approximately 25% of the amount in the wild type. Here, quantitative cytogenetical analysis reveals that the residual chiasmata in Atmsh5 mutants are randomly distributed among meiocytes, suggesting that AtMSH5 has an essential role during interference-sensitive chiasma formation. Taken together, the evidence indicates that AtMSH5 promotes homologous recombination through facilitating chiasma formation during prophase I in Arabidopsis.     

hMSH4-hMSH5 adenosine nucleotide processing and interactions with homologous recombination machinery

We have previously demonstrated that the human heterodimeric meiosis-specific MutS homologs, hMSH4-hMSH5, bind uniquely to a Holliday Junction and its developmental progenitor (Snowden, T., Acharya, S., Butz, C., Berardini, M., and Fishel, R. (2004) Mol. Cell 15, 437-451). ATP binding by hMSH4-hMSH5 resulted in the formation of a sliding clamp that dissociated from the Holliday Junction crossover region embracing two duplex DNA arms. The loading of multiple hMSH4-hMSH5 sliding clamps was anticipated to stabilize the interaction between parental chromosomes during meiosis double-stranded break repair. Here we have identified the interaction region between the individual subunits of hMSH4-hMSH5 that are likely involved in clamp formation and show that each subunit of the heterodimer binds ATP. We have determined that ADP-->ATP exchange is uniquely provoked by Holliday Junction recognition. Moreover, the hydrolysis of ATP by hMSH4-hMSH5 appears to occur after the complex transits the open ends of model Holliday Junction oligonucleotides. Finally, we have identified several components of the double-stranded break repair machinery that strongly interact with hMSH4-hMSH5. These results further underline the function(s) and interactors of hMSH4-hMSH5 that ensure accurate chromosomal repair and segregation during meiosis.     

The Arabidopsis ROCK-N-ROLLERS gene encodes a homolog of the yeast ATP-dependent DNA helicase MER3 and is required for normal meiotic crossover formation

Recent studies in Saccharomyces cerevisiae have unveiled that meiotic recombination crossovers are formed by two genetically distinct pathways: a major interference-sensitive pathway and a minor interference-insensitive pathway. Several proteins, including the MSH4/MSH5 heterodimer and the MER3 DNA helicase, are indispensable for the interference-sensitive pathway. MSH4 homologs have been identified in mice and Arabidopsis and shown to be required for normal levels of crossovers, suggesting that the function of MSH4 may be conserved among major eukaryotic kingdoms. However, it is not known whether an MER3-like function is also required for meiosis in animals and plants. We have identified an Arabidopsis gene that encodes a putative MER3 homolog and is preferentially expressed in meiocytes. T-DNA insertional mutants of this gene exhibit defects in fertility and meiosis. Detailed cytological studies indicate that the mutants are defective in homolog synapsis and crossover formation, resulting in a reduction of bivalents and in the formation of univalents at late prophase I. We have named this gene ROCK-N-ROLLERS (RCK) to reflect the mutant phenotype of chromosomes undergoing the meiotic 'dance' either in pairs or individually. Our results demonstrate that an MER3-like function is required for meiotic crossover in plants and provide further support for the idea that Arabidopsis, like the budding yeast, possesses both interference-sensitive and insensitive pathways for crossover formation.     

Crystal structure and biochemical analysis of the MutS.ADP.beryllium fluoride complex suggests a conserved mechanism for ATP interactions in mismatch repair

During mismatch repair ATP binding and hydrolysis activities by the MutS family proteins are important for both mismatch recognition and for transducing mismatch recognition signals to downstream repair factors. Despite intensive efforts, a MutS.ATP.DNA complex has eluded crystallographic analysis. Searching for ATP analogs that strongly bound to Thermus aquaticus (Taq) MutS, we found that ADP.beryllium fluoride (ABF), acted as a strong inhibitor of several MutS family ATPases. Furthermore, ABF promoted the formation of a ternary complex containing the Saccharomyces cerevisiae MSH2.MSH6 and MLH1.PMS1 proteins bound to mismatch DNA but did not promote dissociation of MSH2.MSH6 from mismatch DNA. Crystallographic analysis of the Taq MutS.DNA.ABF complex indicated that although this complex was very similar to that of MutS.DNA.ADP, both ADP.Mg(2+) moieties in the MutS. DNA.ADP structure were replaced by ABF. Furthermore, a disordered region near the ATP-binding pocket in the MutS B subunit became traceable, whereas the equivalent region in the A subunit that interacts with the mismatched nucleotide remained disordered. Finally, the DNA binding domains of MutS together with the mismatched DNA were shifted upon binding of ABF. We hypothesize that the presence of ABF is communicated between the two MutS subunits through the contact between the ordered loop and Domain III in addition to the intra-subunit helical lever arm that links the ATPase and DNA binding domains.     

Caenorhabditis elegans msh-5 is required for both normal and radiation-induced meiotic crossing over but not for completion of meiosis

Crossing over and chiasma formation during Caenorhabditis elegans meiosis require msh-5, which encodes a conserved germline-specific MutS family member. msh-5 mutant oocytes lack chiasmata between homologous chromosomes, and crossover frequencies are severely reduced in both oocyte and spermatocyte meiosis. Artificially induced DNA breaks do not bypass the requirement for msh-5, suggesting that msh-5 functions after the initiation step of meiotic recombination. msh-5 mutants are apparently competent to repair breaks induced during meiosis, but accomplish repair in a way that does not lead to crossovers between homologs. These results combine with data from budding yeast to establish a conserved role for Msh5 proteins in promoting the crossover outcome of meiotic recombination events. Apart from the crossover deficit, progression through meiotic prophase is largely unperturbed in msh-5 mutants. Homologous chromosomes are fully aligned at the pachytene stage, and germ cells survive to complete meiosis and gametogenesis with high efficiency. Our demonstration that artificially induced breaks generate crossovers and chiasmata using the normal meiotic recombination machinery suggests (1) that association of breaks with a preinitiation complex is not a prerequisite for entering the meiotic recombination pathway and (2) that the decision for a subset of recombination events to become crossovers is made after the initiation step.