Cloning, Structural Characterization, and Chromosomal Localization of the Human Orthologue of Saccharomyces cerevisiae MSH5 Gene Chengtao Her 1 and Norman A. Doggett Life Sciences Division and Center for Human Genome Studies, Mail Stop M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Received February 26, 1998; accepted May 1, 1998 We have cloned and characterized the human ortho- logue of the Saccharomyces cerevisiae MutS homologue 5 (MSH5) cDNA, as well as the human gene that encodes the MSH5 cDNA, as a step toward understanding the molecular genetic mechanisms involved in the biologi- cal function of this novel human protein. The identified cDNA contains a 2505-bp open reading frame (ORF) that encodes an 834-amino-acid polypeptide with a predicted molecular mass of 92.9 kDa. The amino acid sequence encoded by this cDNA includes sequence motifs that are conserved in all known MutS homologues existing in bacteria to humans. The cDNA appears, on the basis of amino acid sequence analysis, to be a member of the MutS family and shares 30% sequence identity with that of S. cerevisiae MSH5, a yeast gene that plays a critical role in facilitating crossover during meiosis. Northern blot analysis demonstrated the presence of a 2.9-kb hu- man MSH5 mRNA species in all human tissues tested, but the highest expression was in human testis, an organ containing cells that undergo constant DNA synthesis and meiosis. The expression pattern of human MSH5 resembled that of the previously identified human MutS homologues MSH2, MSH3, and MSH6— genes that are involved in the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC). In an effort to expedite the search for potential disease association with this new human MutS homologue, we have also determined the chromosomal location and structure of the human MSH5 locus. Sequence and structural characterization demonstrated that MSH5 spans approximately 25 kb and contains 26 exons that range in length from 36 bp for exon 8 to 254 bp for exon 25. MSH5 has been mapped to human chromosome band 6p21.3 by fluorescence in situ hybridization. Knowledge of the sequence and gene structure of MSH5 will now enable studies of the possi- ble roles MSH5 may play in meiosis and/or DNA replica- tive mismatch repair. © 1998 Academic Press INTRODUCTION DNA mismatch repair plays a critical role in main- taining the integrity of genetic information by correct- ing mismatched nucleotides that may occur during DNA synthesis, from the spontaneous deamination of 5-methylcytosine or from errors introduced during ge- netic recombination. The bacterial MutHLS pathway, such as that in Escherichia coli, is the most extensively characterized DNA mismatch repair system (Su and Modrich, 1986; Modrich, 1991; Kolodner, 1996). Recent molecular studies demonstrated that the bacterial MutHLS system was well conserved throughout evolu- tion. From bacteria to humans, mutations in the mis- match repair genes can cause higher mutation rates and increased microsatellite instability (Johnson et al., 1996; Thibodeau et al., 1993). Bacterial MutS homo- logues identified in eukaryotes typically shared 20 to 30% identity in amino acid sequence (Pochart et al., 1997). The budding yeast Saccharomyces cerevisiae has six homologues (MSH1 to MSH6) of the E. coli MutS pro- tein. MSH1 encodes a S. cerevisiae mitochondria- bound protein that appears to play a role in maintain- ing the mitochondria genome (Reenan and Kolodner, 1992). Homologues MSH2 to MSH6 encode proteins that interact with nuclear DNA. These five homo- logues, MSH2 through MSH6, have been shown to have diverse biological functions. The S. cerevisiae MSH2, MSH3, and MSH6 proteins are major compo- nents involved in DNA mismatch repair. The func- tional entity of the S. cerevisiae mismatch repair sys- tem has been demonstrated to be a high-order complex consisting of MSH2–MSH3 and MSH2–MSH6 het- erodimers, in which the MSH2–MSH6 complex recog- nizes both single-base mismatches and small loops formed by insertions or deletions in the DNA; con- versely, the MSH2–MSH3 complex recognizes only small insertions and deletions (Johnson et al., 1996; Marsischky et al., 1996). S. cerevisiae MSH4 and MSH5 play critical roles in meiotic crossover and re- combination but not in DNA mismatch repair (Ross- Macdonald and Roeder, 1994; Hollingsworth et al., Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. AF048986 – AF048991. 1 To whom correspondence and reprint requests should be ad- dressed. Telephone: (505) 665-3358. FAX: (505) 665-3024. E-mail: her@telomere.lanl.gov. GENOMICS 52, 50 – 61 (1998) ARTICLE NO. GE985374 50 0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.