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.     

Human MSH2 (hMSH2) protein controls ATP processing by hMSH2-hMSH6

The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ～8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.     

hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA

Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch. Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular. We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches.     

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.     

Activation of human MutS homologs by 8-oxo-guanine DNA damage.

The DNA lesion 8-oxo-guanine (8-oxo-G) is a highly mutagenic product of the interaction between reactive oxygen species and DNA. To maintain genomic integrity, cells have evolved mechanisms capable of removing this frequently arising oxidative lesion. Mismatch repair (MMR) appears to be one pathway associated with the repair of 8-oxo-G lesions (DeWeese, T. L., Shipman, J. M., Larrier, N. A., Buckley, N. M., Kidd, L. R., Groopman, J. D., Cutler, R. G., te Riele, H., and Nelson, W. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11915-11920; Ni, T. T., Marsischky, G. T., and Kolodner, R. D. (1999) Mol. Cell 4, 439-444). Here we report the effect of double-stranded DNA oligonucleotides containing a single 8-oxo-G on the DNA binding affinity, ATPase, and ADP right arrow ATP exchange activities of hMSH2-hMSH6 and hMSH2-hMSH3. We found that hMSH2-hMSH6 binds the oligonucleotide DNA substrates with the following affinities: 8-oxo-G/T > 8-oxo-G/G > 8-oxo-G/A > 8-oxo-G/C approximately G/C. A similar trend was observed for DNA-stimulated ATPase and ADP --> ATP exchange activities of hMSH2-hMSH6. In contrast, hMSH2-hMSH3 did not appear to bind any of the 8-oxo-G containing DNA substrates nor was there enhanced ATPase or ADP --> ATP exchange activities. These results suggest that only hMSH2-hMSH6 is activated by recognition of 8-oxo-G lesions. Our data are consistent with the notion that post-replication MMR only participates in the repair of mismatched 8-oxo-G lesions.     

The role of mismatched nucleotides in activating the hMSH2-hMSH6 molecular switch

We have previously shown that hMSH2-hMSH6 contains an intrinsic ATPase which is activated by mismatch-provoked ADP-->ATP exchange that coordinately induces the formation of a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone (1,2). These studies suggested that mismatch repair could be propagated by a signaling event transduced via diffusion of ATP-bound hMSH2-hMSH6 molecular switches to the DNA repair machinery. The Molecular Switch model (Fishel, R. (1998) Genes Dev. 12, 2096-2101) is considerably different than the Hydrolysis-Driven Translocation model (Blackwell, L. J., Martik, D., Bjornson, K. P., Bjornson, E. S., and Modrich, P. (1998) J. Biol. Chem. 273, 32055-32062) and makes additional testable predictions beyond the demonstration of hydrolysis-independent diffusion (Gradia, S., Subramanian, D., Wilson, T., Acharya, S., Makhov, A., Griffith, J., and Fishel, R. (1999) Mol. Cell 3, 255-261): (i) individual mismatch-provoked ADP-->ATP exchange should be unique and rate-limiting, and (ii) the k(cat x DNA) for the DNA-stimulated ATPase activity should decrease with increasing chain length. Here we have examined hMSH2-hMSH6 affinity and ATPase stimulatory activity for several DNA substrates containing mispaired nucleotides as well as the chain length dependence of a defined mismatch under physiological conditions. We find that the results are most consistent with the predictions of the Molecular Switch model.     

The effect of O6-methylguanine DNA adducts on the adenosine nucleotide switch functions of hMSH2-hMSH6 and hMSH2-hMSH3

The human homologs of prokaryotic mismatch repair have been shown to mediate the toxicity of certain DNA damaging agents; cells deficient in the mismatch repair pathway exhibit resistance to the killing effects of several of these agents. Although previous studies have suggested that the human MutS homologs, hMSH2-hMSH6, bind to DNA containing a variety of DNA adducts, as well as mispaired nucleotides, a number of studies have suggested that DNA binding does not correlate with repair activity. In contrast, the ability to process adenosine nucleotides by MutS homologs appears to be fundamentally linked to repair activity. In this study, oligonucleotides containing a single well defined O(6)-methylguanine adduct were used to examine the extent of lesion-provoked DNA binding, single-step ADP --> ATP exchange, and steady-state ATPase activity by hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers. Interestingly, O(6)-methylguanine lesions when paired with either a C or T were found to stimulate ADP --> ATP exchange, as well as the ATPase activity of purified hMSH2-hMSH6, whereas there was no significant stimulation of hMSH2-hMSH3. These results suggest that O(6)-methylguanine uniquely activates the molecular switch functions of hMSH2-hMSH6.     

Meiotic recombination: sealing the partnership at the junction

Crossovers ensure proper chromosome segregation in meiosis. A heterodimer of MutS proteins, hMSH4-hMSH5, has recently been found to interact with recombination intermediates in a manner that suggests a mechanism for directing meiotic DNA double strand break repair towards a crossover pathway.     

Dissociation of mismatch recognition and ATPase activity by hMSH2-hMSH3.

MSH2-MSH3 directs the repair of insertion/deletion loops of up to 13 nucleotides in vivo and in vitro. To examine the biochemical basis of this repair specificity, we characterized the mispair binding and ATPase activity of hMSH2-hMSH3. The ATPase was found to be regulated by a mismatch-stimulated ADP --> ATP exchange, which induces a conformational transition by the protein complex. We demonstrated strong binding of hMSH2-hMSH3 to an insertion/deletion loop containing 24 nucleotides that is incapable of provoking ADP --> ATP exchange, suggesting that mismatch recognition appears to be necessary but not sufficient to induce the intrinsic ATPase. These studies support the idea that hMSH2-hMSH3 functions as an adenosine nucleotide-regulated molecular switch that must be activated by mismatched nucleotides for classical mismatch repair to occur.     

Bcl2 impedes DNA mismatch repair by directly regulating the hMSH2-hMSH6 heterodimeric complex

Bcl2 has been reported to suppress DNA mismatch repair (MMR) with promotion of mutagenesis, but the mechanism(s) is not fully understood. MutSalpha is the hMSH2-hMSH6 heterodimer that primarily functions to correct mutations that escape the proofreading activity of DNA polymerase. Here we have discovered that Bcl2 potently suppresses MMR in association with decreased MutSalpha activity and increased mutagenesis. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus, which interacts with hMSH6 but not hMSH2 via its BH4 domain. Deletion of the BH4 domain from Bcl2 abrogates the ability of Bcl2 to interact with hMSH6 and is associated with enhanced MMR efficiency and decreased mutation frequency. Overexpression of Bcl2 reduces formation of the hMSH2-hMSH6 complex in cells, and purified Bcl2 protein directly disrupts the hMSH2-hMSH6 complex and suppresses MMR in vitro. Importantly, depletion of endogenous Bcl2 by RNA interference enhances formation of the hMSH2-hMSH6 complex in association with increased MMR and decreased mutagenesis. Thus, Bcl2 suppression of MMR may occur in a novel mechanism by directly regulating the heterodimeric hMSH2-hMSH6 complex, which potentially contributes to genetic instability and carcinogenesis.     

Intermediates of yeast meiotic recombination contain heteroduplex DNA

The formation of heteroduplex DNA features prominently in all models for homologous recombination. A central intermediate in the current double-strand break repair model contains two Holliday junctions flanking a region of heteroduplex DNA. Studies of yeast meiosis have identified such intermediates but failed to detect associated heteroduplex DNA. We show here that these intermediates contain heteroduplex DNA, providing an important validation of the double-strand break repair model. However, we also detect intermediates where both Holliday junctions are to one side of the initiating DSB site, while the intervening region shows no evidence of heteroduplex DNA. Such structures are not easily accommodated by the canonical version of the double-strand break repair model.     

Interactions of Human hMSH2 with hMSH3 and hMSH2 with hMSH6: Examination of Mutations Found in Hereditary Nonpolyposis Colorectal Cancer

Mutations in the human mismatch repair protein hMSH2 have been found to cosegregate with hereditary nonpolyposis colorectal cancer (HNPCC). Previous biochemical and physical studies have shown that hMSH2 forms specific mispair binding complexes with hMSH3 and hMSH6. We have further characterized these protein interactions by mapping the contact regions within the hMSH2-hMSH3 and the hMSH2-hMSH6 heterodimers. We demonstrate that there are at least two distinct interaction regions of hMSH2 with hMSH3 and hMSH2 with hMSH6. Interestingly, the interaction regions of hMSH2 with either hMSH3 or hMSH6 are identical and there is a coordinated linear orientation of these regions. We examined several missense alterations of hMSH2 found in HNPCC kindreds that are contained within the consensus interaction regions. None of these missense mutations displayed a defect in protein-protein interaction. These data support the notion that these HNPCC-associated mutations may affect some other function of the heterodimeric complexes than simply the static interaction of hMSH2 with hMSH3 or hMSH2 with hMSH6.     

PCNA activates the Holliday junction endonuclease Hjc

The resolving enzyme Hjc, which cleaves Holliday junctions with a high degree of structural specificity, is conserved in all archaea. Like RuvC in Escherichia coli, Hjc functions in the related processes of homologous recombination and double-strand break repair. In bacteria, the RuvAB complex binds Holliday junctions and catalyses ATP-dependent branch migration, but the equivalent proteins in archaea and eukarya are unknown. Here, we demonstrate that Hjc from Sulfolobus solfataricus forms a physical interaction with the sliding clamp PCNA via a C-terminal PCNA-interacting peptide (PIP) motif in Hjc. PCNA stimulates the Holliday junction cleavage activity of Hjc in vitro, and deletion of the PIP motif abrogates this effect. This is the first report of a functional interaction between a sliding clamp and a junction-resolving enzyme, and raises the possibility that PCNA could recruit a variety of different proteins to act on Holliday junctions in vivo.     

Hereditary cancer-associated missense mutations in hMSH6 uncouple ATP hydrolysis from DNA mismatch binding

Hereditary nonpolyposis colorectal cancer is caused by germline mutations in DNA mismatch repair genes. The majority of cases are associated with mutations in hMSH2 or hMLH1; however, about 12% of cases are associated with alterations in hMSH6. The hMSH6 protein forms a heterodimer with hMSH2 that is capable of recognizing a DNA mismatch. The heterodimer then utilizes its adenosine nucleotide processing ability in an, as of yet, unclear mechanism to facilitate communication between the mismatch and a distant strand discrimination site. The majority of reported mutations in hMSH6 are deletions or truncations that entirely eliminate the function of the protein; however, nearly a third of the reported variations are missense mutations whose functional significance is unclear. We analyzed seven cancer-associated single amino acid alterations in hMSH6 distributed throughout the functional domains of the protein to determine their effect on the biochemical activity of the hMSH2-hMSH6 heterodimer. Five alterations affect mismatch-stimulated ATP hydrolysis activity providing functional evidence that missense variants of hMSH6 can disrupt mismatch repair function and may contribute to disease. Of the five mutants that affect mismatch-stimulated ATP hydrolysis, only two (R976H and H1248D) affect mismatch recognition. Thus, three of the mutants (G566R, V878A, and D803G) appear to uncouple the mismatch binding and ATP hydrolysis activities of the heterodimer. We also demonstrate that these three mutations alter ATP-dependent conformation changes of hMSH2-hMSH6, suggesting that cancer-associated mutations in hMSH6 can disrupt the intramolecular signaling that coordinates mismatch binding with adenosine nucleotide processing.     

MSH4 acts in conjunction with MLH1 during mammalian meiosis.

MSH4 is a meiosis-specific MutS homolog. In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast. Germ-line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1-deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals. Here we show that hMSH4 interacts with hMLH1. The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA. The domain of hMSH4 responsible for the interaction is in the amino-terminal part of the protein whereas the region that contains the ATP binding site and helix-turn-helix motif does not bind to hMLH1. Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse. The number of MSH4 foci decreases gradually as pachynema progresses. During this transition, MLH1 foci begin to appear and colocalize with MSH4. These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination.     

A quantitative model of nucleosome dynamics

The expression, replication and repair of eukaryotic genomes require the fundamental organizing unit of chromatin, the nucleosome, to be unwrapped and disassembled. We have developed a quantitative model of nucleosome dynamics which provides a fundamental understanding of these DNA processes. We calibrated this model using results from high precision single molecule nucleosome unzipping experiments, and then tested its predictions for experiments in which nucleosomes are disassembled by the DNA mismatch recognition complex hMSH2-hMSH6. We found that this calibrated model quantitatively describes hMSH2-hMSH6 induced disassembly rates of nucleosomes with two separate DNA sequences and four distinct histone modification states. In addition, this model provides mechanistic insight into nucleosome disassembly by hMSH2-hMSH6 and the influence of histone modifications on this disassembly reaction. This model''s precise agreement with current experiments suggests that it can be applied more generally to provide important mechanistic understanding of the numerous nucleosome alterations that occur during DNA processing.     

Holliday junction binding activity of the human Rad51B protein

The human Rad51B protein is involved in the recombinational repair of damaged DNA. Chromosomal rearrangements of the Rad51B gene have been found in uterine leiomyoma patients, suggesting that the Rad51B gene suppresses tumorigenesis. In the present study, we found that the purified Rad51B protein bound to single-stranded DNA and double-stranded DNA in the presence of ATP and either Mg(2+) or Mn(2+) and hydrolyzed ATP in a DNA-dependent manner. When the synthetic Holliday junction was present along with the half-cruciform and double-stranded oligonucleotides, the Rad51B protein only bound to the synthetic Holliday junction, which mimics a key intermediate in homologous recombination. In contrast, the human Rad51 protein bound to all three DNA substrates with no obvious preference. Therefore, the Rad51B protein may have a specific function in Holliday junction processing in the homologous recombinational repair pathway in humans.     

Mutation in the magnesium binding site of hMSH6 disables the hMutSalpha sliding clamp from translocating along DNA

In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutSalpha, a heterodimer of hMSH2 and hMSH6. In the presence of ATP and magnesium, hMutSalpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp. This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively. We show that a purified recombinant hMutSalpha variant, hMutSalpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with translocation. Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex. The "translocation-prone" conformation of wild type hMutSalpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro. Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gammaS or AMPPNP, respectively. The finding that ATP induces different conformational changes in hMutSalpha in the presence and in the absence of magnesium helps explain the functional differences between hMutSalpha variants incapable of binding ATP as compared with those unable to bind the metal ion.     

N-terminus of hMLH1 confers interaction of hMutLalpha and hMutLbeta with hMutSalpha

Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutSα (hMSH2-hMSH6) and hMutLα (hMLH1-hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutSα recruits hMutLα, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutLα as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutSα using a novel interaction assay. We show that, although the interaction requires ATP, hMutLα does not need to hydrolyse this nucleotide to join hMutSα on DNA, suggesting that ATP hydrolysis by hMutLα happens downstream of complex formation. The analysis of the individual subunits of hMutLα demonstrated that the hMutSα–hMutLα interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N-terminus of hMLH1 confers this interaction. In contrast, only the C-terminus stabilised and co-immunoprecipitated hPMS2 when both proteins were co-expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C-terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutLβ (hMLH1–hPMS1). We show that hMutLβ interacts as efficiently with hMutSα as hMutLα, and that it predominantly binds to hMutSα via hMLH1 as well.     

Interplay between Structure-Specific Endonucleases for Crossover Control during Caenorhabditis elegans Meiosis

The number and distribution of crossover events are tightly regulated at prophase of meiosis I. The resolution of Holliday junctions by structure-specific endonucleases, including MUS-81, SLX-1, XPF-1 and GEN-1, is one of the main mechanisms proposed for crossover formation. However, how these nucleases coordinately resolve Holliday junctions is still unclear. Here we identify both the functional overlap and differences between these four nucleases regarding their roles in crossover formation and control in the Caenorhabditis elegans germline. We show that MUS-81, XPF-1 and SLX-1, but not GEN-1, can bind to HIM-18/SLX4, a key scaffold for nucleases. Analysis of synthetic mitotic defects revealed that MUS-81 and SLX-1, but not XPF-1 and GEN-1, have overlapping roles with the Bloom syndrome helicase ortholog, HIM-6, supporting their in vivo roles in processing recombination intermediates. Taking advantage of the ease of genetic analysis and high-resolution imaging afforded by C. elegans, we examined crossover designation, frequency, distribution and chromosomal morphology in single, double, triple and quadruple mutants of the structure-specific endonucleases. This revealed that XPF-1 functions redundantly with MUS-81 and SLX-1 in executing crossover formation during meiotic double-strand break repair. Analysis of crossover distribution revealed that SLX-1 is required for crossover suppression at the center region of the autosomes. Finally, analysis of chromosome morphology in oocytes at late meiosis I stages uncovered that SLX-1 and XPF-1 promote meiotic chromosomal stability by preventing formation of chromosomal abnormalities. We propose a model in which coordinate action between structure-specific nucleases at different chromosome domains, namely MUS-81, SLX-1 and XPF-1 at the arms and SLX-1 at the center region, exerts positive and negative regulatory roles, respectively, for crossover control during C. elegans meiosis.