ABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , Karyopherins/metabolism , Nuclear Export Signals , Receptors, Cytoplasmic and Nuclear/metabolism , Testis/metabolism , Active Transport, Cell Nucleus/drug effects , Amino Acid Sequence , Animals , Cell Cycle Proteins/analysis , Cell Cycle Proteins/genetics , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cytoplasm/chemistry , Cytoplasm/metabolism , Dimerization , Fatty Acids, Unsaturated/pharmacology , Humans , Karyopherins/genetics , Male , Mice , Mice, Inbred Strains , Mice, Mutant Strains , Molecular Sequence Data , Nuclear Export Signals/genetics , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/genetics , Testis/chemistry , Exportin 1 ProteinABSTRACT
During meiotic prophase, chromosomes must undergo highly regulated recombination events, some of which lead to reciprocal exchanges. In yeast, MSH4, a meiosis-specific homologue of the bacterial MutS protein, is required for meiotic recombination. In mice, disruption of the Msh4 gene results in male and female infertility due to meiotic failure. To date, the implication of MSH4 mutations has not been established in human sterility. However, it is noteworthy that mutant mice exhibit a defect in the chromosome synapsis, strikingly similar to the clinical observations found in human infertility. As a step towards understanding the molecular mechanisms underlying the role of MSH4 in human gametogenesis, we decided to determine whether this protein interacts with recombination machinery enzymes. Our results provide biochemical evidence indicating that the human MSH4 protein physically interacts with both RAD51 and DMC1, two RecA homologues known to initiate DNA strand-exchange between homologous chromosomes. Immunolocalization analyses show that some MSH4 foci, located on mouse meiotic chromosomes, colocalize with DMC1/RAD51 complexes. Our data support the view that MSH4 is associated with the early meiotic recombination machinery in mammals. We consider the possibility that MSH4 is involved in the regulation of recombination events by exerting a function closely after DNA strand-exchange has been initiated.
Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Meiosis/physiology , Animals , Antibodies/immunology , Cell Cycle Proteins/analysis , Cell Cycle Proteins/genetics , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Chromosomes/chemistry , Chromosomes/metabolism , DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , Humans , Immunoprecipitation , Male , Meiosis/genetics , Mice , Nuclear Proteins , Phosphate-Binding Proteins , Rad51 Recombinase , Recombination, Genetic/genetics , Recombination, Genetic/physiology , Spermatocytes/chemistry , Spermatocytes/metabolism , Two-Hybrid System TechniquesABSTRACT
The mismatch-repair (MMR) system plays a central role in maintaining genetic stability and requires evolutionarily conserved protein factors, including MutS and MutL homologs. Since the discovery of a link between the malfunction of post-replicative MMR and human cancers, a number of works have focused on the function of MutS and MutL homologs in the correction of replication errors. However, several MutS-like and MutL-like proteins also participate in meiotic recombination. The MutL homolog MLH3 has been recently identified in mammals. Several pieces of evidence support a role for this protein in post-replicative MMR. To investigate whether MLH3 also acts during meiotic recombination, we analyzed its expression in mammalian germ cells. The MLH3 gene is expressed in mouse meiotic cells and in human testis, and, as revealed by immunoprecipitation assays, the MLH3 protein is found in mouse spermatocytes. We further demonstrate that the meiosis-specific MSH4 protein, known to participate to meiotic recombination, is co-immunoprecipitated with MLH3 from mouse meiotic cell extracts. In addition, the two MLH3 protein isoforms potentially expressed in human testis (hMLH3 and hMLH3 Delta 7) interact in vitro with the hMSH4 protein. These interaction data suggest that MLH3 is associated with MSH4 in mammalian meiotic cells, and strongly support the possibility that MLH3 plays a role in mammalian meiotic recombination.
