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The Role of MutS Homologues MSH4 and MSH5 in DNA Metabolism and Damage Response

The DNA mismatch repair (MMR) pathway is one of the most important genome surveillance systems involved in governing faithful transmission of genetic information during DNA replication and homologous recombination (Jiricny 2006). MMR deficiency attributes to a phenotype known as microsatellite instability (MSI), a condition that predisposes individuals to a heightened risk of cancer development (Harfe & JinksRobertson 2000; Iyer et al. 2006; Jiricny 2006; Kunkel & Erie 2005). Notably, hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome is the most common malignancy identified in individuals with MMR gene mutations. The MMR pathway relies on the coordinated functions of a family of proteins that recognize mismatched nucleotides and initiate subsequent repair actions ranging from excision of a fragment of DNA containing the mismatched nucleotide to DNA repair synthesis. The MMR system is well conserved evolutionarily from bacteria to humans, of which the eukaryotic MMR pathway is evolved to possess multiple homologous genes that carry out conserved and diverse functions corresponding to their bacterial counterparts (Modrich 1991). Genes encoding homologues of the bacterial MMR proteins such as MutS and MutL have been identified in a variety of eukaryotes including yeast, plants, nematodes, and mammals. However, the bacterial MutH appears to be an exception—this methylation-sensitive endonuclease, exclusively functioning in gram-negative bacteria, directs the action of MMR to hemimethylated newly synthesized bacterial DNA (Iyer et al. 2006). All eukaryotic organisms possess multiple MutS homologues—collectively known as MSH proteins—which may number as high as seven (MSH1 to MSH7), although the mitochondrial-localizing MSH1 of Saccharomyces cerevisiae (Reenan & Kolodner 1992) and the Arabadopsis thalia MSH7 (Culligan & Hays 1997) do not appear to be fully conserved (Fishel & Wilson 1997). The functionality of these proteins is similar to that of their counterparts in bacteria such as Eschericia coli. Prokaryotic MutS exists as a homodimer, while eukaryotic MSH proteins form heterodimers in the forms of MSH2-MSH3, MSH2MSH6 and MSH4-MSH5. Two of these eukaryotic heterodimers (MSH2-MSH3 and MSH2-


Introduction
The DNA mismatch repair (MMR) pathway is one of the most important genome surveillance systems involved in governing faithful transmission of genetic information during DNA replication and homologous recombination (Jiricny 2006). MMR deficiency attributes to a phenotype known as microsatellite instability (MSI), a condition that predisposes individuals to a heightened risk of cancer development (Harfe & Jinks-Robertson 2000;Iyer et al. 2006;Jiricny 2006;Kunkel & Erie 2005). Notably, hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome is the most common malignancy identified in individuals with MMR gene mutations. The MMR pathway relies on the coordinated functions of a family of proteins that recognize mismatched nucleotides and initiate subsequent repair actions ranging from excision of a fragment of DNA containing the mismatched nucleotide to DNA repair synthesis. The MMR system is well conserved evolutionarily from bacteria to humans, of which the eukaryotic MMR pathway is evolved to possess multiple homologous genes that carry out conserved and diverse functions corresponding to their bacterial counterparts (Modrich 1991). Genes encoding homologues of the bacterial MMR proteins such as MutS and MutL have been identified in a variety of eukaryotes including yeast, plants, nematodes, and mammals. However, the bacterial MutH appears to be an exception-this methylation-sensitive endonuclease, exclusively functioning in gram-negative bacteria, directs the action of MMR to hemimethylated newly synthesized bacterial DNA (Iyer et al. 2006). All eukaryotic organisms possess multiple MutS homologues-collectively known as MSH proteins-which may number as high as seven (MSH1 to MSH7), although the mitochondrial-localizing MSH1 of Saccharomyces cerevisiae (Reenan & Kolodner 1992) and the Arabadopsis thalia MSH7 (Culligan & Hays 1997) do not appear to be fully conserved (Fishel & Wilson 1997). The functionality of these proteins is similar to that of their counterparts in bacteria such as Eschericia coli. Prokaryotic MutS exists as a homodimer, while eukaryotic MSH proteins form heterodimers in the forms of MSH2-MSH3, MSH2-MSH6 and MSH4-MSH5. Two of these eukaryotic heterodimers (MSH2-MSH3 and MSH2www.intechopen.com MSH6) play fundamental roles in mitotic post-replicative MMR (Fishel & Wilson 1997;Kolodner 1996), in which incorrectly matched bases are replaced with proper partners. In spite of the high levels of sequence homology to the other MSH proteins, what role, if any, of the MutS homologues MSH4 and MSH5 in the process of MMR has yet to be experimentally determined, and the biochemical function(s) of the heterocomplex formed by MSH4 and MSH5 also awaits to be further delineated ). Nevertheless, high levels of expression of both genes in the testes and ovaries implicate a direct role for hMSH4 and hMSH5 in development and meiosis (Bocker et al. 1999;Her & Doggett 1998;Moens et al. 2002). Genetic studies in S. cerevisiae, Caenorhabditis elegans, and Mus musculus highlight an important role for MSH4 and MSH5 in meiotic development of viable gametes, but provide no evidence to substantiate their potential role in the repair of mismatched bases like the other MutS homologues (de Vries et al. 1999;Edelmann et al. 1999;Hollingsworth et al. 1995;Kelly et al. 2000;Kneitz et al. 2000;Zalevsky et al. 1999). The observation that purified recombinant hMSH4-hMSH5 heterocomplex can specifically bind to recombination intermediate structures such as the Holliday junction (Snowden et al. 2004) has implicated a direct role for hMSH4-hMSH5 in the process of meiotic and mitotic recombinational double-strand break (DSB) repair. Gene knockout of Msh4 and/or Msh5 in mice results in defective chromosome synapsis in meiotic prophase I, and therefore sterility-likely attributed to defective homologous recombination (de Vries et al. 1999;Edelmann et al. 1999;Kneitz et al. 2000). Although the expression patterns of MSH4 and MSH5 mRNA in testis support their functional role in meiosis, low levels of MSH4 and MSH5 expression have been identified in many other, non-meiotic organs, and these two genes are not necessarily expressed in concert (de Vries et al. 1999;Edelmann et al. 1999;Her et al. 2001;Her et al. 2003;Her et al. 1999;Kneitz et al. 2000;Paquis-Flucklinger et al. 1997;Winand et al. 1998), implicating MSH4 and/or MSH5 can function in other pathways beyond the scope of meiosis and may function separately as well ). In fact, recent evidence supports the notion that MSH4 and MSH5 are involved in the process of mitotic DNA DSB repair, and may be also involved in other aspects of DNA damage repair and response (Tompkins et al. 2009;Sekine et al. 2007).

Structural and functional features of MSH4 and MSH5
2.1 Gene structure and expression of MSH4 and MSH5 2.1.1 S. cerevisiae MSH4 and MSH5 The identification of MSH4 and MSH5 was first performed in the budding yeast S. cerevisiae, and that led to the classification of these proteins as meiosis-specific members of the MutS homologue family. MSH4 was isolated by a transposon insertion screen resulting in the generation of lacZ fusion genes expressed specifically in meiotic cells (Burns et al. 1994). Following electrophoretic separation of yeast chromosomes, the MSH4 gene was mapped to chromosome VI by Southern blot analysis, located 2 cM from SEC4. Sequence analysis of an MSH4-complementing subclone identified an open reading frame (ORF) of 2634 bp encoding the 878-amino acid MSH4 protein. As a member of the MutS family, MSH4 shows 35% identity to E. coli MutS and 31-36% identity to the other yeast MutS homologues. The similarity is most pronounced in the C-terminal region, which contains the putative ATPbinding domain and helix-turn-helix DNA-binding motif. Despite its extensive homology to bacterial MutS, which functions in the initiation of MMR through direct binding of mispaired bases, MSH4 is meiosis specific. Expression of mutant MSH4 results in defective www.intechopen.com reciprocal recombination and nondisjunction of homologous chromosomes at meiosis I and consequently spore inviability. The MSH4 protein is specifically expressed in meiotic cells where it localizes to discrete chromosomal locations with no apparent involvement in MMR . The MSH5 gene is mapped to chromosome IV, located 1.9 cM from CDC36. The MSH5 ORF is composed of 2703 nucleotides and encodes a protein of 901 amino acids with a predicted molecular mass of 102 kDa (Hollingsworth et al. 1995). Although MSH5 exhibits strong homology to the MutS family of proteins, like MSH4, it is not involved in MMR. Diploids lacking MSH5 display decreased spore viability, increased chromosomal nondisjunction during meiosis I, with a concomitant decrease in reciprocal exchange between-but not within-homologous chromosomes. However, lack of MSH5 expression does not correlate to a decrease in gene conversion. Mutants lacking MSH4 or MSH5 are phenotypically comparable, indicating that these two yeast genes are in the same epistasis group and are likely to serve similar functions (Hollingsworth et al. 1995).

Mouse MSH4 and MSH5
Sequence analysis revealed that the mouse Msh4 ORF is 2874 bp in length with a 196 bp 3'-UTR followed by a poly(A) tract. A polyadenylation signal (AATAAA) is located 24 nucleotides 5' upstream from the poly(A) tract. The 958-amino acid protein encoded by this ORF contains the highly conserved sequence motifs present in all MutS homologues. The cDNA shares 84.1% and 89.3% sequence identities with its human orthologue in nucleotide and amino acid sequences, respectively. The amino terminus of Msh4, on the other hand, is the most divergent and shares no sequence homology with that of the human hMSH4 (Her et al. 2001;Kneitz et al. 2000). Northern blot analysis has indicated that Msh4 is predominantly expressed in the testis, but low levels of expression are also present in the heart, brain, and liver, whereas dot-blot analysis, besides confirming the Northern results, has also revealed low levels of Msh4 expression in several other non-meiotic tissues (Her et al. 2001). The mouse Msh5 gene is located on chromosome 17 in a region that is syntenic to the locus on human chromosome 6 harboring the human gene. The gene for Msh5 contains 24 exons and spans approximately 18 kb with exon length varying from 36 bp for exon 7 to 392 bp for exon 24; intron lengths range from 79 bp for intron 17 to 4687 bp for intron 11. The first 248 nucleotides of exon 1 and the last 133 nucleotides of exon 24 are noncoding. Comparison between the human and mouse homologues reveals that the mouse Msh5 gene shares a high degree of structural homology with that of the human hMSH5 gene. The locations of most exon-intron splicing junctions as well as the lengths of all internal coding exons of Msh5 are identical to that of hMSH5. The mouse Msh5 ORF is 2502 bp in length and encodes an 833amino acid polypeptide with a predicted molecular weight of 92.6 kDa and an isoelectric point of 6.33. Msh5 contains the same set of highly conserved sequence motifs that present in all other MutS proteins (de Vries et al. 1999;Edelmann et al. 1999;Her et al. 1999). Like Msh4, high levels of Msh5 expression are largely confined to the testis while relatively low levels of Msh5 expression are detectable from heart, spleen, liver, and lung (Her et al. 1999). Both male and female mice lacking Msh4 or Msh5 are infertile as a result of meiotic arrest (de Vries et al. 1999;Edelmann et al. 1999;Kneitz et al. 2000). Spermatocyte chromosomes of Msh4 or Msh5 deficient mice do not synapse properly in late zygonema and early pachynema despite DSB formation. Chromosome pairing, normally triggered by meiotic DSB, involves mainly nonhomologous chromosomes in Msh4 or Msh5 deficient mice, of which only a fraction of nuclei in Msh5-null males show partial pairing; while in Msh4-null spermatocyte pairing is typically higher at 70% of all nuclei. As a consequence, germ cells in Msh4 or Msh5 deficient mice fail to enter pachynema and die by apoptosis, leading to testes devoid of post-leptotene spermatocytes. In comparison, female mice deficient in Msh4 or Msh5 suffer similar consequences as in males experiencing pre-pachytene meiotic catastrophe, particularly oocytes become apoptototic prior to birth. In female mice lacking both Msh4 and Msh5, the oocyte pool is completely lost and is accompanied by ovarian degeneration during the first eight to ten weeks of postnatal period (Kneitz et al. 2000).

Human hMSH4 and hMSH5
The human hMSH4 gene is composed of 20 exons and spans 116 Kb on chromosome 1p31. With a 2808 bp ORF, the hMSH4 gene encodes a protein of 936 amino acids with a predicted molecular mass of 104.8 kDa (Paquis-Flucklinger et al. 1997). In contrast, the hMSH5 gene contains 26 exons and spans approximately 25 Kb within the MHC class III region on human chromosome 6p21.3. The hMSH5 gene harbors a 2501 bp ORF encoding an 834amino acid protein with a predicted molecular mass of 92.9 kDa (Her & Doggett 1998;Winand et al. 1998). Moderate levels of hMSH4 transcripts are present in the testis and low levels of hMSH4 transcripts are also detectable in several non-meiotic tissues including the thymus, ovary, colon, pancreas, brain, heart, liver, and placenta (Her et al. 2003;Paquis-Flucklinger et al. 1997). In contrast, the full-length hMSH5 transcripts are detectable in virtually all tissues examined with the most abundant expression in the testis. Northern blot analysis shows the presence of distinct hMSH5 hybridization signals in a variety of tissues such as the thymus, skeletal muscle, bone marrow, spinal cord, brain, trachea, ovary, and lymph node (Bocker et al. 1999;Her and Doggett 1998;Winand et al. 1998). This wide hMSH5 tissue distribution pattern has been also recapitulated in RT-PCR analysis of various human tissues and is well reflected in the human EST database. The difference in the expression patterns of these two genes raises the possibility that hMSH4 and hMSH5 may function independently of one another in certain tissue types beyond the meiotic arena ).

Splicing variants and nonsynonymous polymorphisms of hMSH4 and hMSH5 in humans
Recent evidence substantiates the existence of multiple alternatively spliced transcripts for hMSH4 and hMSH5 in human cells (Table 1). Although the functional significance of these alternatively spliced transcripts is presently unknown, the expression profiles and some properties of the proteins encoded by the splicing variants are being characterized. For instance, one hMSH4 splicing variant, hMSH4sv, resulting from exon 19-skipping, has been analyzed. hMSH4sv harbors a frameshift of 7 amino acids followed by a stop codon in exon 20, thus producing an 850-amino acid polypeptide. Truncated at the carboxyl terminal, the hMSH4sv contains most of the conserved sequence motifs presented in all MutS homologues except for the carboxyl terminal helix-turn-helix motif (Her et al. 2003). The tissue distribution profile of hMSH4sv is similar but not identical to that of hMSH4, suggesting that hMSH4sv might be subjected to differential regulation in some tissue types including the heart, liver, placenta, and ovary (Her et al. 2003). Intriguingly, the protein encoded by hMSH4sv is incapable of interacting with hMSH5, but it does interact with von www.intechopen.com Hippel-Lindau tumor suppressor-binding protein 1(VBP1) (Her et al. 2003). Also of note is another hMSH4 exon-skipping variant (hMSH4) that lacks the entire exon 6 leading to an in-frame deletion of hMSH4 amino acid residues 273 to 330 (Her et al. 2003;Santucci-Darmanin et al. 1999). The hMSH4 homodimerization domain, residing in between amino acid residues 148 and 387 (Her et al. 2003), significantly overlaps with the region encoded by hMSH4 exon 6, thus hMSH4 is expected to be defective in homotypic interaction. The hMSH5 gene produces multiple alternative transcripts, of which four hMSH5 variants that maintain the reading frame have been identified (hMSH5a, hMSH5b, hMSH5c, and hMSH5d; UniGene database) and hMSH5c appears to be identical to that of the originally described human hMSH5. Referenced by the deduced amino acid sequence of hMSH5, it is evident that hMSH5a (hMSH5sv) encodes an 851-amino acid protein containing a 17-amino acid insertion between codons 179 and 180, owing to the retention of the last 51-bp of hMSH5 intron 6 (Yi et al. 2005), whereas hMSH5b harbors one extra amino acid residue between codons 654 and 655-due to the retention of the last 3 nucleotides of hMSH5 intron 20 (Her & Doggett 1998). hMSH5d represents the shortest hMSH5 variant. Although it contains the 17-amino acid insertion and the one extra amino acid residue described above, hMSH5d lacks 30 amino acid residues corresponding to codons 744 to 773. The existence of different hMSH5 variant transcripts keenly supports the possibility that hMSH5 may encode multiple products; a thorough understanding of the functional aspects of these potential protein isoforms requires detailed experimental examination of these variants. In particular, it is necessary to determine whether these hMSH5 variants are resulted from sequence variations within the corresponding introns, or they are created by a yet-to-be-defined mechanism. Presently, besides hMSH5, only one splicing variant, hMSH5sv, has been experimentally analyzed (Yi et al. 2005). The expression profile of hMSH5sv appears to be distinguishable from that of hMSH5; for example, the expression of hMSH5sv, but not hMSH5, is absent or below the detection limit in the brain, heart, and skeletal muscle. In addition, the expression of hMSH5sv displays a large variation in tumor cell lines with breast and lung carcinomas showing the most abundant expression. In contrast to hMSH4sv, displaying impaired interaction with hMSH5, hMSH5sv has maintained its capacity to interact with hMSH4 (Yi et al. 2005). In addition to the existence of multiple alternatively spliced transcripts, potential diverse functions involved with the hMSH4 and hMSH5 genes are also being reflected by the fact that both genes are associated with many coding region single nucleotide polymorphisms (SNPs), of which many are non-synonymous. There are at least seven non-synonymous SNPs that have been identified for each of the genes (Table 1). For hMSH4 and hMSH5, the corresponding single amino acid changes caused by these SNPs are hMSH4 A60V, A90T, A97T, E162K, I365V, Y589C, S914N, and hMSH5 P29S, L85F, Y202C, V206F, R351G, L377F, P786S. However, the allele frequencies, haplotypes, and functional implications of most, if not all, SNPs are largely undetermined; in fact only one non-synonymous SNP (rs2075789), hMSH5 C85T (hMSH5 P29S ), has been characterized experimentally as a common genetic polymorphism with an allele frequency of 11.6% in an American Caucasian population of 99 individuals and 17% in a Chinese population of 279 individuals Yi et al. 2005). Located within the hMSH5 amino terminal proline-rich interacting domain for hMSH4 and c-Abl, the Pro 29 to Ser alteration causes a moderate reduction of protein interaction with hMSH4, whereas this alteration promotes the activation of c-Abl kinase activity and therefore enhances ionizing radiation (IR) induced p73-dependent apoptosis www.intechopen.com

Variants and nonsynonymous polymorphisms
Changes in amino acid (aa) residues hMSH4 hMSH4sv Exon 19 skipping leading to the production of a 850 aa polypeptide, of which the last 7 aa are frame-shifted hMSH4 Exon 6 (Tompkins et al. 2009;Yi et al. 2006;Yi et al. 2005). Given the essential role of Msh5 in ovarian and testicular development in mice (de Vries et al. 1999;Edelmann et al. 1999), it is interesting to note that the hMSH5 C85T SNP is relatively enriched in ovarian cancer patients and is associated with a higher risk for azoospermia or severe oligozoospermia in humans (Xu et al. 2010;Yi et al. 2005). Another noteworthy hMSH5 variant is hMSH5 L85F/P786S -encoded by an hMSH5 allele harboring two co-segregating SNPs (C253T and C2356T)-that displays compromised ability to interact with hMSH4. Intriguingly, the allele for hMSH5 L85F/P786S has been associated with the occurrence of human immunoglobulin deficiency syndromes, i.e. IgA deficiency (IgAD) and common variable immune deficiency (CVID) (Sekine et al. 2007). It is also important to note that two hMSH5 noncoding SNPs have been linked to conditions in humans as well. A recent genome-wide association study (GWAS) of 511,919 SNPs in populations with Caucasian origin has identified a high risk factor for lung cancer development within the hMSH5 gene locus at 6p21.33 (Wang et al. 2008). Specifically, a significant increase in lung cancer susceptibility is associated with rs3131379, a SNP located within intron 10 of the hMSH5 gene. Another study, designed to identify genetic markers for the adverse reaction associated with the use of Allopurinol-a common medication for gout and hyperuricemia, has revealed a tight link with the hMSH5 locus (Hung et al. 2005). The evidence from this study demonstrates a significant association of a separate hMSH5 SNP (rs1150793) with the risk of developing severe cutaneous adverse reactions (SCAR) in Han Chinese patients treated with Allopurinol.
Although the existence of multiple hMSH4 and hMSH5 splicing variants and various SNPs poses a daunting task for a thorough appreciation of their functions, close analysis of their properties at molecular and cellular levels, especially for those with clinical significance, would be necessary for delineating the mechanistic basis underlying their potential link to disease conditions in humans. It is highly plausible that functional effects similar to those observed for hMSH5 P29S could also be conferred by other hMSH5 and/or hMSH4 nonsynonymous SNPs. It should not be a surprise that different combinations of these SNPs might associate with an array of subtle functional alterations; that, to a certain extent, could also affect the dynamic interplay among hMSH4-hMSH5 associated proteins and subsequent downstream events.

Structural properties of MSH4 and MSH5 proteins 2.2.1 General molecular structure of MutS homologues
A common characteristic of MutS homologous proteins is their essential role in binding and recognizing mismatched base pairs-a function facilitated by their ability to act as DNAbinding ATPases. Whilst structures of eukaryotic MSH proteins remains undetermined, insights towards the mechanistic aspects of mismatch recognition may be inferred from the crystal structure of bacterial MutS protein. Crystallographic studies of homologues in T. aquaticus and E. coli suggest that the MutS homodimer binds to heteroduplex DNA during mismatch recognition as a "structural heterodimer" (Junop et al. 2001;Lamers et al. 2000;Obmolova et al. 2000). The homodimerization of MutS protein is mediated through the region harboring MutS ATPase activity. The dimerization domain is far apart from the DNA binding domain, but these regions coordinate through conformational changes triggered by MutS binding to heteroduplex DNA or ATP (Lamers et al. 2004;Lamers et al. 2003). In essence, these studies indicate that MutS is a modular protein with separate domains which, when dimerized at their carboxyl termini, act to encircle mismatch-containing DNA during the initiation stages of repair process. Deletion analysis of MutS protein confirms that the carboxyl terminus, which also includes a P-loop motif for nucleoside triphosphate binding, is involved in homodimerization. The amino terminal end is necessary for binding to mismatch-containing DNA, and through the binding and hydrolysis of ATP in the carboxyl terminal, MutS may dissociate from mismatched DNA once repair is underway. Although no crystallographic analysis of eukaryotic MSH proteins is presently available, noting the high degree of homology between MutS homologues and their bacterial counterparts, it is not difficult to conjecture that the MSH proteins found in yeast, mouse, and humans may contain similar structural features to those of prokaryotic MutS proteins.

Eukaryotic MSH4-MSH5 complex
Whereas functional bacterial MutS protein exists as homodimers, the eukaryotic MSH family is far more complex and contains multiple heterodimers composed of different MSH www.intechopen.com proteins. MSH heterodimers carry out diverse cellular functions including MMR and DNA damage response. However, MSH4 and MSH5 are unique in their intracellular roles. Although they contain the conserved sequence motifs found in all MutS homologues-such as the ATP binding domain and a helix-turn-helix structural motif located at the carboxyl terminal half of the protein (Burns et al. 1994;Her & Doggett 1998;Her et al. 2001;Her et al. 1999;Hollingsworth et al. 1995;Paquis-Flucklinger et al. 1997)-neither MSH4 nor MSH5 interact with the other MSH proteins known to function in MMR. MSH4 and MSH5 interact with each other exclusively, forming a distinctive heterocomplex (Bocker et al. 1999;Her et al. 2001;Her et al. 1999;Winand et al. 1998;Yi et al. 2005). Unlike other MSH proteins, which contain specific amino acid residues for recognition and binding of mismatched base pairs, MSH4 and MSH5, either individually or as a heterocomplex, are unable to detect or respond to mismatches as they lack structural motifs for binding of mismatched base pairs (Obmolova et al. 2000). It is postulated that the MSH4-MSH5 heterocomplex may configure in a way that can accommodate large recombination intermediate DNA structures such as Holliday junction intermediates (Obmolova et al. 2000). Evidence obtained with the purified recombinant hMSH4-hMSH5 heterocomplex points to their role in the recognition and binding of artificial DNA structures resembling four-way junctions (Snowden et al. 2004), suggesting that the hMSH4-hMSH5 heterocomplex is uniquely equipped for processing recombinational intermediates. The human hMSH4 and hMSH5 interact with one another via the carboxyl terminal region of hMSH4 and both the amino and carboxyl terminal regions of hMSH5. The first 109 and the last 103 amino acid residues of hMSH5 are necessary for the formation of a composite hMSH4-interacting domain; however, only the carboxyl terminal 93 amino acid residues of hMSH4 are required to interact with hMSH5 (Yi et al. 2005), suggesting an asymmetric structural partition of hMSH4 and hMSH5 in the heterocomplex. The hMSH4-hMSH5 heterocomplex is suggested to form a sliding clamp structure that stabilizes and preserves Holliday junctions during prophase of meiosis I or in the repair of DSBs. A model for hMSH4-hMSH5 in meiotic recombination has been proposed (Snowden et al. 2004), implicating their role in linking DSB repair to the regulation of crossover (CO) formation. It is known that the amino terminal region of hMSH4, composed of amino acid residues 148-387, is involved in mediating homotypic interaction (Her et al. 1999;Lee et al. 2006). Due to the physical separation of the hetero-and homo-interacting domains on hMSH4, it has been suggested that hMSH4 and hMSH5 may form a multimeric protein complex such as a tetramer. In addition, it has been demonstrated that the interface of hMSH4-hMSH5 heterocomplex forms a composite interaction domain for GPS2; the latter is a protein factor involved in intracellular signaling and DNA damage response (Jin et al. 1997;Lee et al. 2006;Peng et al. 2001;Spain et al. 1996). The interplay of GPS2 with the hMSH4-hMSH5 heterocomplex may provide a link to downstream molecular events required for Holliday junction processing and subsequent resolution.

Protein interacting partners of MSH4 and MSH5
The MSH4-MSH5 heterocomplex is thought to participate in a limited array of functions, leaving the individual proteins to coordinate specific cellular processes independent of one another throughout various mammalian tissues. It has been shown that hMSH4 physically interacts with hMLH1 as well as its binding partner hMLH3, in which the amino terminal region of hMSH4 interacts with hMLH1 proteolytic degradation products, rather than the full-length hMLH1 (Lipkin et al. 2000;Santucci-Darmanin et al. 2002;Santucci-Darmanin et www.intechopen.com al. 2000). Furthermore, hMSH4 interacts with VBP1, Rad51, and DMC1 (Her et al. 2003;Her et al. 2007;Neyton et al. 2004). In addition to its interacting partner hMSH4, hMSH5 has been shown to interact with the non-receptor tyrosine kinase c-Abl, hMRE11, and histone demethylase SMCY (Akimoto et al. 2008;Kato et al. 2007;Yi et al. 2006), of which the interaction with c-Abl mediates hMSH5 tyrosine phosphorylation in response to IR-induced DSBs . Coherent with this observation, the ubiquitous DNA damage repair protein hRad51 coexists in the protein complex containing both hMSH5 and c-Abl .
Interaction with different protein partners may provide a foundation for hMSH4 and hMSH5 to act independently in specific cellular processes, during which protein interactions can also modulate the functions of hMSH4 and hMSH5. For example, the interaction of VBP1 with hMSH4 negatively regulates the formation of hMSH4-hMSH5 heterocomplex (Her et al. 2003). The biological relevance of this observation in mitotic cells is presently not known. It is reported that, during early stages of mouse testis development, the upregulation of GPS2 coincides with the down-regulation of VBP1 immediately prior to, or at the onset of, the first meiotic wave, presumably facilitating the formation of Msh4-Msh5 heterocomplex . The hMSH4-hMSH5 interaction is also subjected to regulation by c-Abl-mediated hMSH5 tyrosine phosphorylation. In particular, hMSH5 is shown to undergo IR-induced c-Abl-dependent tyrosine phosphorylation, and consequently this posttranslational modification leads to the dissociation of hMSH4-hMSH5 heterocomplex (Tompkins et al. 2009;Yi et al. 2006). Since the formation of hMSH4-hMSH5 heterocomplex is required for the interaction with GPS2 (likely in a complex with HDAC3), it is expected that hMSH5 tyrosine phosphorylation will result in a dynamic transformation of the hMSH4-hMSH5 associated protein complex, which might be functionally required during recombinational DSB repair. Recent evidence suggests that factors influence the hMSH4-hMSH5 interaction will also affect their subcellular localization. It appears that hMSH4-hMSH5 dimerization enhances their nuclear localization-possibly facilitated by a nuclear localization signal (NLS) located in the middle of hMSH5 protein, or by masking of a CRM1-dependent nuclear export signal (NES) on the carboxyl terminal region of hMSH5 within the hMSH4-interacting domain (Lahaye et al. 2010;Neyton et al. 2007).

Functions of MSH4 and MSH5 2.3.1 Role in meiotic recombination
Meiotic recombination occurs in meiotic prophase starting with the formation of programmed DSBs induced by the expression of SPO11, a protein highly conserved and enriched in germ cells at recombination hotspots (Baudat & de Massy 2007;Keeney et al. 1997). After 5' end resection at the break, the emerging 3' single-stranded overhang invades undamaged homologous chromosome or sister chromatid, leading to the formation of meiotic recombination intermediate structures including the Holliday junction and thus facilitating DSB repair (Szostak et al. 1983). The completion of meiotic homologous recombination is achieved through the resolution of Holliday junction structures by cleavage and rejoining to re-form two separate DNA molecules. The two major outcomes of meiotic homologous recombinational DSB repair are chromosomal reciprocal exchange or CO and gene conversion or non-crossover (Mahadevaiah et al. 2001). While non-crossover is important for conserving genetic identities, chromosomal CO is crucial for creating genetic diversity and therefore promoting survival along the way of evolution. Furthermore, COs www.intechopen.com are critical for successful meiosis, of which both the frequency and the distribution of COs are well controlled in a way that governs the fidelity of chromosome segregation (Youds & Boulton 2011). Studies carried out in budding yeast have provided evidence to indicate that both MSH4 and MSH5 are involved in meiotic recombination but not in MMR (Hollingsworth et al. 1995;. MSH4-null mutation in S. cerevisiae is not associated with elevated rate in mismatches of reporter genes Can R (Reenan & Kolodner 1992) and Thr+ (Kramer et al. 1989) or defective gene conversion, instead the MSH4-null strain displays 2 to 3-fold reduction in CO and an increase in homologous chromosome nondisjunction, leading to reduced spore viability (Ross-Macdonald & Roeder 1994). Similar effects are evident in S. cerevisiae with mutant MSH5, in which MMR is proficient in the processes of both meiosis and mitosis, and meiotic gene conversion frequency is not significantly different from the wild type. However, CO is reduced 2 to 3-fold and homologous chromosome nondisjunction is elevated in meiosis I. As a consequence, spore viability is reduced to 37% from 72% observed in the wild type (Hollingsworth et al. 1995). S. cerevisiae strain lacking both MSH4 and MSH5 does not show a synergistic effect on CO frequency in meiosis I, and the spore viability is compatible with either mutants, indicating MSH4 and MSH5 act in the same pathway that facilitate meiotic crossing over recombination (Hollingsworth et al. 1995). Decreased crossover interference and delayed or incomplete chromosome synapsis are also observed in MSH4 mutant yeast, suggesting a role for MSH4 in the regulation of crossover distribution (Novak et al. 2001). Like budding yeast, C. elegans MSH4 and MSH5 are functionally conserved with respect to their role in promoting meiotic CO. With a null mutation in him-14, the C. elegans ortholog of MSH4, CO is abolished during meiosis I in both oocytes and spermatocytes. As a result, formation of chiasmata is absent and chromosome segregation is severely defective. Most of the him-14 embryos, although produced in normal amounts, fail to hatch; and and among the hatched that reached to adulthood, 45% are males (Zalevsky et al. 1999;Zetka & Rose 1995). This is consistent with an increase in non-disjunction, because males are normally arisen through spontaneous non-disjunction with a frequency of about 0.1% in the hermaphrodite germ line. The him-14 mutants, however, do not show any abnormality in meiotic chromosome pairing and synapsis (Zalevsky et al. 1999). Similar to their budding yeast counterparts, the mutant worms do not display increased "mutator" phenotype, assayed by the means of levamisole resistance, indicating that Him-14 is not required for MMR in C. elegans (Zalevsky et al. 1999). In spite of high degree of conservation, C. elegans and S. cerevisiae MSH4 mutants differ significantly in their effects on CO. Without MSH4, CO is completely abrogated in C. elegans (Zalevsky et al. 1999), yet about 30-50% is retained in the budding yeast . This difference has helped to fashion the current view that C. elegans has a single or a dominant pathway to create meiotic CO for which MSH4 is indispensible, and budding yeast has alternative MSH4-independent pathway(s) for CO generation. While MSH4 is crucial for proper chromosome segregation, in contrast to yeast, C. elegans Him-14 does not appear to act on chromosome pairing and synapsis. Likewise germ cells from C. elegans msh-5 mutants are able to progress through meiosis with normal chromosome pairing and synapsis. Msh-5 mutants lay eggs at a same rate as the wild type, and they produce a normal number of embryos, however, more than 97% of the embryos fail to hatch and 42% of those survived to adulthood are males, owing to defective segregation of homologous chromosomes (Kelly et al. 2000). Similar to the him-14 mutants, CO frequency in Msh-5 mutants is reduced to about 1% of the wild type, and these mutants www.intechopen.com lack chiasmata formation (Kelly et al. 2000). On the basis of their nearly identical effect on meiotic recombination, it is conceivable that both MSH4 and MSH5 are indispensible for C. elegans meiotic CO, and these two MutS homologues act in the same process through apparently the only pathway in this species. In mice, meiotic recombination is also initiated by Spo11-dependent DSBs (Mahadevaiah et al. 2001). Msh5 knockout mice are generally healthy and undergo normal development, however their reproductive organs do not develop properly. For example, testes in Msh5null animals are significantly smaller in size and ovaries quickly become rudimentary after birth (de Vries et al. 1999;Edelmann et al. 1999). Although their mating behavior appears normal, Msh5-null mice are sterile. Histological analysis demonstrates that the testes of Msh5-null mice are completely devoid of epididymal spermatozoa, presumably due to apoptosis before pachytene-the stage of meiosis when crossover structures between homologous chromosomes become experimentally visible (de Vries et al. 1999;. Closer examination of Msh5-null spermatocytes and oocytes reveals disrupted chromosome pairing and impaired synaptonemal complex formation, indicating Msh5 is essential for homologous chromosome pairing and synapsis during meiosis I in mice (de Vries et al. 1999;Edelmann et al. 1999). Since the single strand DNA binding protein Rad51 typically represents a marker for recombination initiation (Bishop 1994;Moens et al. 2002), the observed Rad51 foci formation on unsynapsed chromosomes in Msh5-null mice tends to suggest that Msh5 is not involved in the initiation of meiotic recombination . Similarly, Msh4-null mice develop normally but are infertile. These mutant mice display severe abnormality in chromosome pairing in early prophase I (Kneitz et al. 2000), indicating Msh4 is also required for meiotic homologous chromosome pairing. However, Msh4 deficiency appears to cause less severe defect in chromosome pairing in comparison to Msh5-null animals. Approximately 69% of nuclei in Msh4-null spermatocytes have some degree of chromosome pairing, but less than 10% of the nuclei contain very few paired chromosomes in Msh5-null mice (Kneitz et al. 2000). Meiotic phenotypes of Msh4 and Msh5 double-knockout mice are similar to the Msh5-null mice, suggesting that Msh4 and Msh5 act together in promoting meiotic chromosome pairing and synapsis in the early phase of meiotic recombination, possibly with Msh5 upstream of Msh4 (Kneitz et al. 2000).
In human oocytes, both hMSH4 and hMSH5 proteins are localized on meiotic chromosomes throughout meiotic prophase I and become co-localized at zygonema with SYCP3, a key component of synaptonemal complex, thus supporting the idea that human hMSH4 and hMSH5 may possess meiotic properties similar to their mouse counterparts (Lenzi et al. 2005). This view is further supported by the physical interaction observed between hMSH4 and hRad51 as well as the co-localization of their counterparts in mouse spermatocytes (Neyton et al. 2004)-indicative of an early role for MSH4, presumably for MSH5 as well, in meiotic recombination. The presence of hMSH4 and hMSH5 foci on meiotic chromosomes after synaptonemal complex formation is highly suggestive of an additional role for these proteins in the late stages of meiotic recombination (Lenzi et al. 2005). Consistent with this speculation is the observed in vitro physical interaction between hMSH4 and hMLH1, the later is commonly regarded as a marker for crossing over recombination (Lynn et al. 2004).
Together with the co-localization of these two proteins in mouse spermatocytes (Santucci-Darmanin et al. 2000), it is suggested that MSH4 and MSH5 play at least two separate roles in meiosis I-an early role in homology searching that leads to proper chromosome pairing and a late role in the processing of recombination intermediate structures. Binding of www.intechopen.com purified hMSH4-hMSH5 heterocomplex to the core of the Holliday junction intermediate structures has provided in vitro evidence to support the late role of these two proteins. It is hypothesized that the binding of progenitor Holliday junction DNA provokes ATP binding to both hMSH4 and hMSH5 in the heterocomplex, and the loading of hMSH4-hMSH5 is projected to stabilize and preserve meiotic recombination intermediates prior to proper resolution (Snowden et al. 2004). I n s h o r t , b o t h M S H 4 a n d M S H 5 p l a y i m p o rtant roles in meiotic recombination. The similarity in phenotypes among MSH4-null, MSH5-null, and MSH4-null/MSH5-null organisms suggests that these two MutS proteins act in the same pathways and likely function as a heterocomplex at least during certain phases of the recombination process.
Although MSH4 and MSH5 homologues in lower eukaryotes and mammals appear to share conserved properties supporting their common function in meiosis, their precise effects diverge in different species. For instance, the major action of MSH4 and MSH5 in lower eukaryotes is on the generation and control of COs, whereas in mammals these two MutS homologues have evolved to exert an early role in governing homologous chromosome pairing and synapsis.

Role in mitotic recombination and DNA damage response
The most described function of MMR proteins is the repair of base-pairing errors arising from DNA replication or recombination. Defects in MMR proteins lead to mutations and MSI. Mutations in MMR genes are known to cause Lynch syndrome (or HNPCC) and to increase the risk and progression of a wide-variety of sporadic cancers. During MMR, replication errors are recognized either by MutS, a heterodimer of MSH2 and MSH6 that binds to single base-base mismatches or small insertion-deletion loops, or by MutS, a heterodimer of MSH2 and MSH3 that binds to larger insertion-deletion loops (Jiricny 2006). The repair process then proceeds with the recruitment of a MutL activity (i.e. one of these three complexes: MLH1-PMS2, MLH1-PMS1 or MLH1-MLH3) and exonucleases to facilitate subsequent excision followed by DNA repair synthesis. The MMR proteins are frequently recognized as major players in mediating cellular responses to DNA damage. As components of the BRCA-1-associated genome surveillance complex , MMR proteins are thought to mediate DNA damage response through either direct sensing of various DNA distortions or functioning as transducers to couple damage detection and the control of cell cycle progression (Jiricny 2006). For instance, MMR proteins are involved in provoking a G2/M phase cell cycle arrest that is vital for both DNA repair and cell death by apoptosis, especially for cells carrying excessive DNA lesions. To this end, it is important to note that MMR deficiency has been linked to DNA damage tolerance, and MMR deficient cells are found to be frequently resistant to killing by various chemotherapeutics including alkylating agents and antimetabolites. Given that resistance to chemotherapeutic agents is of a great concern in cancer treatment, a thorough understanding of the molecular mechanisms involved with MMR in cellular DNA damage response will undoubtedly pave a way for devising more effective therapeutic strategies.
Although MSH4 and MSH5 have not been indicated experimentally to function in the process of MMR, cumulating evidence has pointed to their potential involvement in mitotic DNA repair and damage response in addition to their role in meiosis. The discrepant expression patterns of MSH4 and MSH5 in mammalian tissues have suggested that these two proteins may function independently of one another and may operate beyond meiosis www.intechopen.com Yi et al. 2005). Studies in yeast have raised the possibility that MSH5 may be involved in cellular response to DNA damage induced by alkylating agents. Specifically, a mutant MSH5 allele (i.e. msh5-14, encoding MSH5 Y823H ) but not MSH5-null mutant in the yeast strain XS-14 (lacking O 6 -methylguanine methyltransferase) confers cellular tolerance to alkylating compound N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Bawa and Xiao 1997;. Although the exact mechanism of action remains to be explored, these studies do indicate that this gain-of-function MSH5 mutant is involved in mediating mitotic cellular response to DNA damage. It is evident that many of the proteins involved in meiotic recombination are also important players in mitotic recombinational DSB repair. Thus, the involvement of MSH5 in meiotic recombination is suggestive of a potential role for MSH5 in mitotic recombinational DSB repair. DSBs are considered to be the most lethal form of DNA lesion that may arise from replication fork collapse, exposure to DNA damaging agents, or programmed cellular processes including the initiation of meiotic recombination in germ cells, class-switch recombination (CSR), and V(D)J recombination in lymphocytes (Ataian & Krebs 2006). In response to DSB formation, dividing cells usually undergo G2/M phase arrest, then either repair the lesion and resume cell cycle or enter the path of apoptosis depending on the extent of damage. Cells lacking proper G2/M cell cycle arrest, apoptosis, or damage repair are often at a higher risk for malignant transformation. The repair of DSBs requires either the non-homologous end joining (NHEJ) or the homologous recombination pathways. While NHEJ is a rapid means utilized by somatic cells to repair DSBs, it is error-prone and can result in alteration of DNA sequences for non-compatible breaks (Dery & Masson 2007;Helleday et al. 2007). On the other hand, homologous recombination is a more accurate repair pathway during S/G2 phases as it utilizes the homologous template usually provided by a sister chromatid or homologous chromosome (Saleh-Gohari & Helleday 2004). Similar to meiotic homologous recombination, this homology-directed DSB repair will also produce two alternative outcomes, CO or non-crossover/gene conversion. Loss of essential homologous recombination gene products often results in chromosome instability, by which cells exhibit increased sensitivities to a variety of DNA damage agents such as IR, cisplatin, and gemcitabine. In addition, homologous recombination deficient cells may also display DNA damage tolerance and resistance to killing by DNA damaging agents as well (Crul et al. 2003;Khanna & Jackson 2001;Takata et al. 2001;van Waardenburg et al. 2004;Zdraveski et al. 2000). As supported by the observed interactions with hRad51, c-Abl, and hMRE11 as well as structures resembling Holliday junctions Kato et al. 2007;Neyton et al. 2004;Snowden et al. 2004;Yi et al. 2006), hMSH4 and hMSH5 are expected to play functional roles in mitotic DNA damage response. In C. elegans oocytes, silencing of RAD51 on a MSH5 deficient background results in chromosome fragmentation, while a comparable defect is also noted when MSH4 and BRCA1 homologues are concomitantly silenced, indicating the existence of functional interplay of these proteins in the maintenance of chromosome integrity (Adamo et al. 2008;Rinaldo et al. 2002). The interaction between hMSH5 and c-Abl can provoke two different cellular actions depending on the severity of DNA damage (Tompkins et al. 2009). Endogenous hMSH5 protein undergoes IR dose and time dependent induction, and it appears that increased levels of hMSH5 can promote IR-triggered apoptosis. However, this effect is more prominent in cells treated with a relatively high dose of IR (> 2 Gy). This is reminiscent of a previous observation that the linear correlation between DSB repair and the number of H2Ax foci only exists in cells irradiated with IR at www.intechopen.com doses below 2 Gy (Bouquet et al. 2006), suggesting the existence of a dynamic regulatory mechanism controlling DSB repair and apoptosis. Present evidence is compatible with the view that the expression of hMSH5 is normally maintained at a low level in unperturbed cells, and DNA damage-triggered hMSH5 induction promotes c-Abl activation and subsequent initiation of a p73-mediated caspase 3-dependent apoptotic response (Tompkins et al. 2009). Coherently, the peculiar interaction between hMSH5 P29S and c-Abl is capable of over-activating c-Abl, leading to increased cellular radiosensitivity. In contrast, moderate hMSH5 induction is expected to facilitate recombinational DSB repair. Recent experimental results have demonstrated a functional requirement for c-Abl-mediated hMSH5 phosphorylation in DSB repair Tompkins et al. 2009). It is demonstrated, by the use of a chromosomally integrated recombination reporter, that hMSH5 tyrosine phosphorylation is an essential early event for non-crossover DSB repair. In fact, cells harboring a phosphorylation deficient hMSH5 mutant are more sensitive to DSB-inducing anticancer drug cisplatin. Collectively, these studies tend to suggest a dual role for the induction of hMSH5 protein in recombinational DSB repair and DNA damage-triggered apoptotic response.

Role in immunoglobulin diversification
Genomic rearrangement that occurs during B-cell development in the form of V(D)J recombination is essential for the generation of antibody repertoire, and additional diversity is achieved by elevated mutation rate and gene sequence shuffling in corresponding processes known as somatic hypermutation (SHM) and CSR (Li et al. 2004b;Schroeder & Cavacini 2010). While SHM targets the "hot spots" in variable (V) region of immunoglobulin (Ig) to enhance antigen recognition, CSR is responsible for Ig isotype switching by way of recombining the switch (S) regions upstream of each functional genes (C H ) in the constant (C) region of Ig heavy (H) chain, i.e. switching from IgM or IgD to other isotypes that mediate diverse effector functions in the daughter cells (Li et al. 2004b;Schroeder & Cavacini 2010). Both SHM and CSR start from deamination of dC to dU by activation-induced cytidine deaminase, a centroblast B-cell specific protein (Muramatsu et al. 2000;Muramatsu et al. 1999). Deamination of dCs leads to G-U mismatches and may subsequently generate C to T or G to A mutations. In addition, uracil N-glycosylase, apurinic/apyrimidinic endonucleases, and MMR proteins are required to generate extended single base mutations and single-stranded DNA nicks (Di Noia & Neuberger 2002;Ehrenstein & Neuberger 1999;Ehrenstein et al. 2001;Guikema et al. 2007;Imai et al. 2003;Martin & Scharff 2002;Rada et al. 2002;Schrader et al. 1999). During CSR, DSBs have to be generated in the S regions, possibly by way of single-strand breaks, to allow intrachromosomal deletion via recombination between the two S regions (Wuerffel et al. 1997), in which DSBs are primarily repaired by NHEJ. The tandem repeats of short consensus sequences in the S regions are too short for homologous recombination, however they are enough for microhomology-mediated end joining (MMEJ), a specified type of NHEJ using small homologous regions to anneal the two overhangs at the break site. It is generally accepted that MMEJ represents a prominent process of NHEJ in CSR (Kenter 1999), and the length of microhomology at S-S junctions may change due to impaired MMEJ (Stavnezer 2000). It is well documented that proteins involved in MMR and DSB repair are also involved in the process of CSR. In fact, Msh2, Msh6, Mlh1, and Pms2 knockout mice all exhibit impaired CSR and SHM (Ehrenstein & Neuberger 1999;Martin & Scharff 2002;Schrader et al. 1999). Mice deficient for early DSB response factors ATM or H2AX, and NHEJ proteins Ku70, Ku80, or DNA-PKcs, show reduced CSR activities but with normal SHM (Bemark et al. 2000;Casellas et al. 1998;Manis et al. 2002;Manis et al. 1998;Reina-San-Martin et al. 2004;Reina-San-Martin et al. 2003). MutS and MutL homologues are not only required to assist MMEJ, but they also play overlapping and distinct roles in the process of switch recombination (Ehrenstein et al. 2001;Li et al. 2004a;Schrader et al. 1999;Schrader et al. 2002). This assessment is mainly obtained through analyzing microhomology at the S-S junctions as well as the distribution of breakpoints in the absence of each protein (Ehrenstein et al. 2001;Li et al. 2004a;Schrader et al. 1999;Wu et al. 2006). Collectively, deficiency of MutS and MutL homologues has been associated with three different phenotypes at the S-S junctions-Msh2 or Mlh3 deficiency leads to a decrease in the length of microhomology (Schrader et al. 2002;Wu et al. 2006), Msh6-null B cells show no change of microhomology (Li et al. 2004a), and an increase in the length of microhomology is evident in Mlh1-or Pms2-null B cells (Ehrenstein et al. 2001;Schrader et al. 2002). MLH1-PMS2 acts downstream of MSH2-MSH6 during MMR, similar effect would be anticipated if the process of MMR played a predominant role. The presence of different microhomology phenotypes indicates that these MMR proteins are involved, at least in part, in different sub-pathways during MMEJ. Indeed, PMS2 has been speculated to interact and stabilize MMEJ intermediates, whereas MSH2 might participate in DNA end processing (Ehrenstein et al. 2001;Schrader et al. 1999;Schrader et al. 2002). Present evidence suggests that MSH4 and MSH5 are additional players in CSR. These two MutS homologues are known to function in the early and late steps of meiotic recombination as well as in mitotic DNA damage response-events that share some similarities to crucial steps in the complex CSR process. Transcripts of hMSH5 are present in human spleen and peripheral blood B cells (Her & Doggett 1998;Sekine et al. 2007).
Association studies have linked one of the non-synonymous alleles of hMSH5 to IgAD and CVID, two syndromes that are attributable to abnormal CSR. The allele encoding the hMSH5 L85F/P786S variant is significantly more frequent in IgAD patients and with borderline significance in CVID patients. One hMSH5 SNP (rs3131378), located within intron 12, is also tightly associated with IgAD and CVID. Furthermore, CVID patients carrying the hMSH5 L85F/P786S allele display increased microhomology at S-S1 joints, which commonly associate with far fewer mutations (Sekine et al. 2007). The L85F and P786S alterations, located within the hMSH4-interacting domain, significantly compromise the protein interaction between hMSH5 and hMSH4, suggesting both hMSH5 and hMSH4 are required for efficient CSR (Sekine et al. 2007). In mice, however, the effects of Msh5 on CSR seem to be strain-specific. In an earlier study, most of the congenic H-2 b/b MRL/Ipr mice (introgression of H-2 b MHC haplotype from129/Sv onto a MRL/Ipr background) show undetectable serum IgG3, reduced levels of serum IgA and elevated levels of serum IgM and IgG2a in older mice, suggesting impaired CSR in these animals. Microarray analysis of gene expression reveals a hypomorphic allele of Msh5 on the H-2 b haplotype. Comparing to controls, H-2 b/b MRL/Ipr mice without serum IgG3 (IgG3 neg ) exhibit significantly increased microhomology at S-S3 (i.e. IgM to IgG3 switch) and S-S (i.e. IgM to IgA switch) joints in splenic B cells (Sekine et al. 2007). This phenotype of increased microhomology is also observed in B cells from Msh5-null FVB mice (de Vries et al. 1999) and Msh4 knockout mice on C57BL/6 background (Sekine et al. 2007).
These studies indicate that both MSH5 and MSH4 are involved in CSR and may function as a heterodimer. Interestingly, studies performed with Msh5-null C57BL/6 mice show no detectable alterations in the length of microhomology (Guikema et al. 2008). It is conceivable that the different effects of Msh5 deficiency on CSR in these two mouse strains might be attributable to different levels of Msh5 expression, of which MRL/lpr is a high Msh5 www.intechopen.com expresser, whereas C57BL/6 expresses very low levels of Msh5, and the difference between them is about 100-fold (Sekine et al. 2007;Sekine et al. 2009). It is also important to note that these two Msh5-null mouse lines have been reported to display different degrees of meiotic chromosome pairing defects (de Vries et al. 1999;Edelmann et al. 1999), suggesting that the role of Msh5 might be influenced by its associated genetic backgrounds.

Conclusion
The MMR system has received a considerable amount of attention over the last decade. It is now clear that the MMR system is multifaceted and participates in several different pathways of DNA metabolism. Among all MMR components, the MutS family of proteins plays critical and conserved functions during the initiation phase of mismatch recognition. In contrast to those MutS homologues involved in MMR, the functions of MSH4 and MSH5 are still not fully understood. These two MutS homologues share similar structure and sequence features with the other members of the MutS family. Present evidence suggests that MSH4 and MSH5 have evolved to function in recombinational DSB repair, DNA damage signaling, and immunoglobulin class switch recombination. Although the mechanistic details of their involvement in these processes have yet to be elucidated, it is conceivable that the actions of hMSH5 in recombinational DSB repair is both hMSH4independent and hMSH4-dependent, presumably coordinating with their functions in homology searching and the resolution of recombination intermediate structures. Available evidence supports a scenario that the expression of hMSH5 is maintained at a low level under normal conditions, whereas DNA damage-elicited hMSH5 induction can promote c-Abl activation and the initiation of a p73-mediated caspase 3-dependent apoptotic response. On the contrary, moderate hMSH5 induction, caused by less severe DNA damage, is expected to facilitate recombinational DSB repair. It is plausible that hMSH4 and hMSH5 may also play a role in CSR, in particular, through manipulating the process of MMEJ. The current and emerging evidence has lent support to the idea that MSH4 and MSH5 are involved in diverse functions by engaging different pathways through various interactions with different proteins. Undoubtedly, the current knowledge about these two MutS homologues has created a solid steppingstone for future exploration of their biological functions and potential association with disease conditions in humans.