[CANCER RESEARCH 59, 816 – 822, February 15, 1999]
`
`Advances in Brief
`
`hMSH5: A Human MutS Homologue That Forms a Novel Heterodimer with
`hMSH4 and Is Expressed during Spermatogenesis1
`
`Tina Bocker, Alan Barusevicius, Tim Snowden, Debora Rasio, Shawn Guerrette, David Robbins, Carl Schmidt,
`John Burczak, Carlo M. Croce, Terry Copeland, Albert J. Kovatich, and Richard Fishel2
`Departments of Microbiology and Immunology [T. B., T. S., D. R., S. G., C. M. C., R. F.] and Pathology and Cell Biology [A. B., A. J. K.], Kimmel Cancer Institute, Thomas
`Jefferson University and Medical College, Philadelphia, Pennsylvania 19107; SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406 [D. R., C. S., J. B.];
`and ABL-Basic Research Program, Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [T. C.].
`
`Abstract
`
`MutS homologues have been identified in nearly all organisms exam-
`ined to date. They play essential roles in maintaining mitotic genetic
`fidelity and meiotic segregation fidelity. MutS homologues appear to
`function as a molecular switch that signals genomic manipulation events.
`Here we describe the identification of the human homologue of the Sac-
`charomyces cerevisiae MSH5, which is known to participate in meiotic
`segregation fidelity and crossing-over. The human MSH5 (hMSH5) was
`localized to chromosome 6p22-21 and appears to play a role in meiosis
`because expression is induced during spermatogenesis between the late
`primary spermatocytes and the elongated spermatid phase. hMSH5 in-
`teracts specifically with hMSH4, confirming the generality of functional
`heterodimeric interactions in the eukaryotic MutS homologue, which also
`includes hMSH2-hMSH3 and hMSH2-hMSH6.
`
`Introduction
`
`The eukaryotic MutS homologues fall into two categories: those
`involved in mismatch repair, and those that are involved in meiotic
`recombination processes (reviewed in Ref. 1). In humans and in yeast,
`the interaction between MSH2 with MSH3 and MSH6, as well as their
`mismatch binding properties, have been studied extensively (2–7).
`hMSH2 copurifies and physically interacts with hMSH3 or hMSH6
`(5–7), and these heterodimers possess overlapping and redundant
`mismatch binding activities with respect to the type of mismatch they
`recognize (7). Moreover,
`the hMSH2-hMSH6 complex has been
`shown to function as a molecular switch that binds to DNA mis-
`matches in the presence of ADP and is then released from the
`mismatch when the ADP is exchanged for ATP (8). The hydrolysis of
`ATP by hMSH2-hMSH6 results in recovery of mispair binding ac-
`tivity (8).
`Germ-line mutations of hMSH2, hMSH6, hMLH1, and hPMS2
`result in a common cancer syndrome, hereditary non-polyposis colo-
`rectal cancer (Lynch syndrome), where predisposition to colorectal,
`endometrial, and other neoplasms is inherited in a dominant pattern
`(9 –13). hPMS1 has also been reported to predispose to colorectal
`cancer (11). However, with the exception of a single apparent germ-
`line mutation, there does not appear to be further evidence of its
`involvement in colorectal or any other cancers. Furthermore, the
`nearest yeast homologues to hPMS1 are MLH2 and MLH3, which
`appear to play little or no role in mismatch repair, and mice deleted for
`PMS1 are not predisposed to develop tumors (14, 15). Thus, the role
`
`Received 7/17/98; accepted 1/5/99.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`1 This work was supported by Grants CA56542 and CA67007 (to R. F.). T. B. was
`supported by Grant Bo/1445-2 from the Deutsche Forschungsgemeinschaft.
`2 To whom requests for reprints should be addressed, at Thomas Jefferson University,
`Jefferson Medical College, Kimmel Cancer Institute, 233 South 10th Street, Philadelphia, PA
`19107. Phone (215) 503-1345; Fax: (215) 923-1098; E-mail: rfishel@hendrix.jci.tju.edu.
`
`of hPMS1 appears to be significantly different from hMSH2, hMSH3,
`hMSH6, hMLH1, and hPMS2.
`Recently, another human MutS homologue, hMSH4, has been
`identified (16). High levels of hMSH4 transcript were found in testis,
`whereas significantly lower levels were found in ovary. No distinctive
`hybridization signal was obtained in any other tissues tested. This
`finding appears to reflect the function of the Saccharomyces cerevi-
`siae MSH4, which is specific for meiosis, is associated with chromo-
`somes during pachytene, and appears to facilitate crossing-overs (17).
`Thus, mutation of msh4 in S. cerevisiae leads to homologous nondis-
`junction in meiosis I and spore inviability. However, the yeast msh4
`does not display any mismatch repair defects in either vegetative or
`meiotic cells (17). Interestingly, the S. cerevisiae MSH4 protein
`appears to form a heterodimeric complex with another yeast MutS
`homologue, MSH5 (18), and this interaction has been shown to be
`insensitive to alteration of the consensus adenine nucleotide binding
`domain. Furthermore, neither MSH4 nor MSH5 interacts with MSH2
`or MSH6, suggesting that MSH4 and MSH5 constitute a class of
`MutS homologue that are functionally different from the proteins that
`participate in mismatch repair (18).
`The identification of a human MSH5 was published while the
`manuscript was under review (19). Here, we have also identified the
`human MSH5 gene (hMSH5)3 and demonstrate its interaction with
`hMSH4, but not with hMSH2, hMSH3, or hMSH6. We additionally
`show a high level of transcript in testis and immunohistochemical
`expression of the hMSH5 protein during a phase of spermatogenesis
`starting after early primary spermatocytes and ending with elongated
`spermatids. These results suggest that hMSH5 may play a role in the
`development of germ cells.
`
`Materials and Methods
`
`Cloning the hMSH4 and hMSH5 cDNAs. A search of the National
`Center for Biotechnology Information EST database revealed a 466-bp se-
`quence derived from Soares human fetal liver spleen cDNA (T67203) with
`strong homology to both yeast MSH3 and MSH5. In parallel, the amino acid
`sequence from the yeast and human mismatch repair proteins MSH2 was used
`to screen the Human Genome Sciences computer database with the TFASTA
`computer software designed by the Genetics Computer Group (University of
`Wisconsin). The Human Genome Sciences database contains nucleotide se-
`quence information of ESTs4 (20), which identify a diverse collection of
`cDNAs derived from more than 400 cDNA libraries. One EST (designated C4)
`was found to have significant homology but not identity to the yeast and
`human MSH2 and MSH3 protein sequence. We amplified two PCR fragments
`using primers derived from these two EST sequences from human testis cDNA
`and used the PCR product to screen a normal testis cDNA library (Clontech,
`Palo Alto, CA) by conventional plaque hybridization. One of these primer sets
`
`3 Sequence data from have been deposited with the GenBank Data Library under
`Accession Number AF034759.
`4 The abbreviations used are: EST, expressed sequence tag; IVTT, in vitro transcription
`and translation; IHC, immunohistochemistry; GST, glutathione S-transferase.
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`hMSH5, A MEIOSIS-SPECIFIC HUMAN MutS HOMOLOGUE
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`(derived from C4) gave a consistent sequence and identified numerous phage
`clones (Primers: forward, 59-ACG CCA TCT TCA CAC GAA T-39; reverse,
`59-TGC AGT GGC ATT GTT CAC T-39). Six positive clones were identified
`and excised via the pDR2 phagemid according to the manufacturer’s recom-
`mendations. Double strand sequencing of the six clones, subcloned into pBSK
`(Stratagene, La Jolla, CA), was performed with the PRISM Ready Reaction
`DyeDeoxy Terminator Cycle Sequencing kit on an Applied Biosystems 377
`sequencer (Foster City, CA). One clone, b29, contained an open reading frame
`of 2505 bp with one STOP codon NH2-terminal to the start methionine and one
`STOP codon at the COOH terminus. The entirety of the NH2 terminus was
`further confirmed with a rapid amplification of cDNA ends reaction performed
`on human normal testis cDNA (Clontech) as described earlier (21). The EST
`sequence obtained from National Center for Biotechnology Information
`(T67203) was found to be contained in the COOH-terminal portion of the b29
`clone. Clone b29 was further subcloned into pGEX (Pharmacia, Piscataway,
`NJ) for the expression of the GST fusion protein in Escherichia coli XL1 Blue
`(Stratagene, La Jolla, CA) and into pET29a (Clontech) for IVTT with NdeI and
`NotI (New England Biolabs, Beverley, MA).
`The hMSH4 clone was obtained from a human testis cDNA (Clontech) by
`PCR with subsequent ligation into the pCR2.1 vector (TA cloning kit; Invitro-
`gen, San Diego, CA). Primer sequences were: outer PCR: forward, 59-GGA
`AGG TTT GGG AGG ATG CTG AGG-39; reverse, 59-ATT GTG ATT ATT
`CTT CAG TCT T-39; nested PCR: forward, 59-ATC TCG AGA TGC TGA
`GGC CTG AG-39; reverse, 59-GCG CTA GCT TAT TCT TCA GTC TTT
`TC-39. The hMSH4 clone was confirmed by complete double strand sequenc-
`ing of both strands and found to contain a deletion of a C in codon 18 and an
`insertion of a G incodon 20, resulting in a V19S and V20S, as well as a G 3
`A at nucleotide 1219, resulting in an E407K amino acid substitution compared
`to the published sequence (numbered starting with the A in the ATG initiator
`codon). The sequences found in the original report were never obtained from
`several different template cDNAs. In addition, we have found an apparent
`polymorphism at codon 368 (CGC 3 AGA) that does not alter the coding Arg.
`Chromosomal Mapping of hMSH5. PCR reactions were performed
`using the primers described above, respectively, to screen the Genebridge4
`Radiation Hybrid Panel (22). Thirty-five cycles were performed with an
`annealing temperature of 60°C for 30 s, followed by 72°C for 1 min. Frag-
`ments were visualized by agarose gel electrophoresis, and data were submitted
`to the Whitehead Institute/MIT Center for Genome Research for final analysis.
`Northern Blotting. Three multiple tissue Northern blots containing poly
`A1 RNA of a total of 23 different human tissues were purchased from
`Clontech. Fifty ng of a full-length hMSH5 cDNA and a b-actin cDNA control
`were radiolabeled with [a-32P] dCTP by random primed labeling (Boehringer
`Mannheim, Mannheim, Germany), and the Northern Blots were hybridized
`according to the manufacturer’s instructions. Alternatively, a 596-bp fragment
`was obtained by PCR with the forward primer 59-CTG GAC GTC ATT CAG
`TTT and the reverse primer 59-CAG CTC CTT GGT TCG GGC ACT ACG-39
`and used as a probe. The blots were washed in 0.3 M NaCl-30 mM sodium
`citrate, pH 7.0; 0.05% SDS at room temperature for a total of 60 min, and at
`50°C in 15 mM NaCl-1.5 mM sodium citrate, pH 7.0; 0.1% SDS for a total of
`40 min. PhosphoImager screens were exposed for 1 day. A 2.5–2.6-kb tran-
`script was detected at a high level in testis. Tissues with significantly lower
`expression levels are bone marrow, lymph nodes, ovary, brain, and spinal cord.
`Antibodies. Five different 15-mer peptides were synthesized that corre-
`spond to predicted immunogenic regions of the hMSH5 protein and conjugated
`to hemocyanin; polyclonal antibodies were raised in rabbits (H.T.I. Bio-
`Products, Ramona, CA). Clone C934-2 was found to be most sensitive and
`specific in Western Blot experiments and was purified over a protein A column
`for Western analysis. Further affinity purification of the antibody was per-
`formed using a crude lysate of Sf9 insect cells overexpressing hMSH5 protein.
`hMSH5 protein lysate was separated by SDS-PAGE and transferred to nitro-
`cellulose, and the hMSH5-specific region was excised and used to affinity
`purify the antibody (23).
`Immunohistochemistry. Sections (5 mm) of formalin-fixed and paraffin-
`embedded tissues were cut onto Neoprene-coated slides (Aldrich Chemicals,
`Milwaukee, WI). After deparaffinization including a 30-min Methanolic per-
`oxide block for endogenous peroxidase activity (Leica Autostainer, Leica,
`Deerfield, IL), the slides were microwaved in 200 ml of Chem.Mate H.I.E.R
`buffer, pH 5.5–5.7 (Ventana Medical Systems, Tucson, AZ) at high energy for
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`5 min (Panasonic Microwave NN-5602A, Franklin PK, IL). Fifty ml of H2O
`were replaced for an additional microwaving step of 4 min at high energy.
`Immunostaining with the catalyzed signal amplification system (DAKO,
`Carpinteria, CA) was performed according to the manufacturer’s instructions
`and incubation with protein A, and hMSH5 specific affinity-purified poly-
`clonal antibody took 50 min at room temperature at a concentration of 1:800
`or 1:2000 with the hMSH2 polyclonal antibody (Ab-3; Oncogene Research
`Products, Cambridge, MA), respectively. For counterstaining with Harris
`Hematoxylin (Surgipath, Richmond, IL), the Leica Autostainer was used.
`GST Fusion Protein Interaction Assay. Five hundred ml of 5 ml of
`overnight starter cultures of pGEX-Fusion proteins with hMSH2, hMSH3,
`hMSH4, hMSH5, and hMSH6, as well as pGEX without insert (negative
`control), were inoculated in 50 ml of Luria broth with 50 mg/ml ampicillin and
`grown to an A600 of 0.5. Protein expression was induced with 0.1 mM
`isopropyl-1-thio-b-D-galactopyranoside for 2 h at 30°C. The cells were pel-
`leted and resuspended in 750 ml of PBS containing protease inhibitors. A
`10-min digestion on ice with 1 mg/ml Lysozyme followed. After the addition
`of 0.2% Triton X-100 and 1 mM DTT, the lysate was snap frozen in liquid
`nitrogen and thawed twice. A DNAseI digest was performed (Boehringer
`Mannheim) at a concentration of 200 units/ml for 30 min on ice, and the cell
`debris was spun down at 14,000 rpm at 4°C for 30 min. Equal amounts of the
`lysates of the different fusion proteins or with GST alone as a negative control
`were incubated on a rocking platform for 1 h at 4°C with 2 mg of glutathione-
`agarose beads (Sigma Chemical Co., St. Louis, MO) each, which had been
`preswollen in PBS with proteinase inhibitors for 1 h at room temperature. The
`beads were washed three times with 500 ml of interaction buffer [20 mM Tris z
`HCl (pH 7.5), 10% glycerol, 150 mM NaCl, 0.1% Tween 20, 5 mM EDTA, 1
`mM DTT, 0.75 mg/ml BSA (Amresco, Solon, OH), and proteinase inhibitors]
`and subsequently incubated in interaction buffer for 1 h at 4°C on a rocking
`platform.
`IVTTs were performed on 1 mg of each hMSH2, hMSH3, hMSH5, and
`hMSH6 inserts in pET vectors and on hMSH4 in pCR 2.1 with the TNT-
`coupled reticulocyte lysate system (Promega Corp., Madison, WI) according to
`the manufacturer’s protocol incorporating 40 mCi of [35S]methionine. Five ml
`of the IVTTs were added to 500 ml of beads in interaction buffer and incubated
`for 1 h at 4°C on the rocking platform. After three final washing steps, the
`supernatant was removed carefully, and the beads were resuspended in 35 ml
`of 23 Spear’s, boiled for 5 min, and spun for 5 min at 14000 rpm. Fifteen ml
`of each reaction were loaded on an 8% SDS-PAGE Gel (Bio-Rad MiniProtean
`II) and run for about 90 min at 135 V. Molecular Dynamics PhosphoImager
`screens were exposed to the dried gels for 1 day.
`
`Results
`
`Isolation and Chromosomal Mapping of hMSH5, a New Hu-
`man MutS Homologue. A total of six positive clones using se-
`quences derived from an EST database were obtained, and both
`strands were sequenced completely. The sequence analysis of clone
`b29 showed an open reading frame of 2505 bp coding for a putative
`834-amino acid protein (Fig. 1) with a predicted Mr 97,000. NH2-
`terminal to the Start-ATG was one STOP codon, and the entirety of
`the sequence was further confirmed by 59-rapid amplification of
`cDNA ends. The nucleotide sequence differs from the report pub-
`lished previously (19) because we found a C 3 T at nucleotide
`position 2292, which would result in a leucine (CTT) instead of a
`valine (CCT) in the amino acid sequence.
`Using the primer sequences described (see “Materials and Meth-
`ods”), we screened the Genebridge4 Radiation Hybrid Panel for PCR
`products of the appropriate size. Submission of the data to Whitehead
`Institute revealed that the hMSH5 gene was located 6.94cR from
`D6S478 on chromosome 6p22.1-21.3.
`MSH5 Defines a New Family of MutS Homologue involved in
`Sporulation and Meiosis. Of all eukaryotic and prokaryotic MutS
`homologues, the b29 clone was found to be most closely related to
`ceMSH5 (29% identity) and scMSH5 (25% identity) with a region
`encompassing the adenine nucleotide binding domain displaying
`;60% identity between these homologues. Thus, the gene was called
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`hMSH5, A MEIOSIS-SPECIFIC HUMAN MutS HOMOLOGUE
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`Fig. 1. Nucleotide sequence and predicted amino acid sequence of hMSH5.
`
`tides derived from the hMSH5 protein sequence. One of those
`hMSH5. In the family of MutS homologues, the next closest relatives
`(C934-2) displayed high specificity toward hMSH5 expressed in Sf9
`are the MSH2 cousins, whereas hMSH3 and hMSH6 appear to be
`cells from a recombinant baculovirus (Fig. 3B). Western blot analysis
`derived from a second branch of the human MutS homologue (Fig.
`revealed expression of hMSH5 in testis and tonsil tissue and at very
`2A) and more closely related to the bacterial MutS proteins. In the
`low levels in two T- and B-cell tumor lines (Jurkat, CEM, Daudi, and
`present alignment, the MSH4 cousins appear the most divergent.
`GM1500; data not shown). It is important to note that the Western
`Interestingly, there is a cohort of MutS homologues found in a subset
`signal in these tissues contained several low molecular weight protein
`of bacteria that are homologous to hMSH5 but largely unrelated to
`band(s) that were also found when a number of secondary detection
`their postreplication mismatch repair cousins. There do not appear to
`systems were used alone. These results suggested that the low mo-
`be any distinguishing characteristics between these bacteria that
`lecular weight bands not identifiable as hMSH5 were the result of
`would clearly shed light on the function of these MutS homologues
`nonspecific interaction between the secondary detection systems and
`and the MSH5 proteins. As with other MutS homologues, the most
`that a high resolution secondary detection system was required to
`highly conserved region surrounds the adenine nucleotide binding
`enhance specificity of the antibody. Such a system was developed for
`domain, although the MSH5 cousins appear to be the most divergent
`immunohistochemical studies (see “Materials and Methods”), but the
`(Fig. 2B).
`Expression of hMSH5. A high level of hMSH5 transcript was
`same system is not amenable for use in Western analysis. The pres-
`ence of hMSH5 transcript in tissues where B- and T-cells develop as
`found in the testis (Fig. 3A). These results appear to correspond to the
`well as expression in the T- and B-cell lines may suggest a relation-
`finding in yeast, where MSH5 appeared meiosis specific (24). The
`ship to other cellular development processes that may also include
`size of the transcript corresponds well to the length of the cDNA
`recombination events. However, it is also possible that the low levels
`sequence, which is 2.5 kb. hMSH5 transcript expression was also
`of hMSH5 protein expression in the B- and T-cell lines could result
`observed in bone marrow, lymph node, brain, spinal cord, trachea, and
`from the fact that the cell lines are derived from hematological
`ovary (Fig. 3A).
`malignancies and thus do not represent normal B- and T-cell precur-
`Several polyclonal antibodies were developed from specific pep-
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`hMSH5, A MEIOSIS-SPECIFIC HUMAN MutS HOMOLOGUE
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`strong nuclear positivity in spermatids in statu nascendi, within round
`and elongated spermatids (S3), whereas all the phases of spermato-
`genesis up to early primary spermatocytes as well as the spermatozoa
`themselves were completely negative (Fig. 4, A–D). This observation
`suggests that hMSH5 plays a specific role in the processes associated
`with the late first or the second meiotic division (Fig. 4I). Because the
`testicular histology of the surgical orchiectomy specimens was not
`entirely normal, we cannot rule out abnormal expression of hMSH5 in
`these testicular samples. In the samples shown, histological examina-
`tion reveals the presence of discrete lymphocytic infiltrates and oc-
`casional intratubular neoplasia (scattered single tumor cells in the
`tubules that are characterized by a pale large cytoplasm and a large
`round nucleus). However, spermatogenesis in these samples appeared
`to be functioning sufficiently to produce mature sperm cells, and a
`number of tubules were found where there was no evidence of
`neoplasia (Fig. 4A). Furthermore, staining of spermatids is evident in
`all of the tubules that appear largely normal based on the presence of
`all stages of spermatogenesis. Textbook examples of normal tubules
`would show the cell types of spermatogenesis filling the entire tubule.
`However, entirely normal fresh testis tissue could not be obtained, and
`IHC was not possible in autopsy material because of widespread
`protein degradation.
`In contrast, hMSH2 is expressed in the nuclei at nearly all phases
`of spermatogenesis except for the round and elongated spermatids
`(where hMSH5 is expressed) and the spermatozoa, which are negative
`(Fig. 4E). The Sertoli cells exhibit a very faint nuclear staining with
`the hMSH2 antibody (Fig. 4E). The hMSH2 expression in tissue is
`clearly correlated with proliferation in general, which is exemplified
`in this study by the strong nuclear positivity in the seminoma (Fig.
`4F). In addition, tissues that were positive for hMSH2 were also
`positive for the proliferation marker Ki67 (data not shown). hMSH5
`protein expression is completely negative in the seminoma as well as
`in other testicular malignancies (e.g., embryonal cell carcinoma, ma-
`ture and immature teratoma; data not shown; Fig. 4G). Note that
`expression of hMSH5 is absent in dividing spermatogonium A (see M,
`Fig. 4D), suggesting that expression is not induced during mitosis.
`Protein Interaction Studies. Because the human MutS homo-
`logue hMSH2, hMSH3, and hMSH6 are known to act as het-
`erodimers,
`interaction studies of hMSH5 with hMSH2, hMSH3,
`hMSH4, and hMSH6 were performed. In these studies, GST-fusion
`proteins (GST) containing hMSH(x) “bait” were incubated with 35S-
`labeled IVTT hMSH(y) “prey.” Specific interactions were detected
`as labeled proteins that precipitated with the GST-hMSH(x) when
`glutathione-agarose beads were introduced. As positive controls, we
`demonstrate that hMSH2 interacts strongly with hMSH3 either as an
`IVTT-hMSH2 with GST-hMSH3 (Fig. 5A) or a GST-hMSH2 with
`IVTT-hMSH3 (Fig. 5B). Similarly, hMSH2 strongly interacts with
`hMSH6, either as an IVTT-hMSH2 and GST-hMSH6 (data not
`shown, Ref. 7) or as a GST-hMSH2 and IVTT-hMSH6 (Fig. 5E). The
`negative controls are lysates expressing the GST moiety alone, which
`
`Fig. 2. MutS homologues. A, family tree. The abbreviations of the different organisms
`are given in alphabetical order: aa, Aquifex aeolicus; ap, Aquifex pyrophilicus; at,
`Arabidopsis thaliana; av, Azotobacter vinelandii; bs, Bacillus subtilis; ce, Caenorhabditis
`elegans; dm, Drosophila melanogaster; ec, Escherichia coli; h, Homo sapiens; hi, Hae-
`mophilus influenzae type b; hp, Helicobacter pylori; mm, Mus musculus; nc, Neurospora
`crassa; rn, Rattus norvegicus; sc, Saccharomyces cerevisiae; sp, S. pombe; spn, Strepto-
`coccus pneumoniae; st, Salmonella typhimurium; sy, Synechocystis sp.; ta, Thermus
`aquaticus; tm, Thermotoga maritima; tt, Thermus thermophilus. B, conservation of the
`adenosine nucleotide binding domain of the known MSH5 homologues.
`
`sors or other undefined factors. Fresh bone marrow from a healthy
`person for Western analysis or immunohistochemistry could not be
`obtained for these studies. The presence of hMSH5 transcript in brain,
`spinal cord, and trachea is unclear.
`Western analysis suggested that the purified polyclonal antibody
`C934-2 derived from a synthetic peptide might be useful in immuno-
`histochemical (IHC) studies. For these studies, we used catalyzed
`signal amplification (see “Materials and Methods”). Specificity was
`determined by comparing samples prepared by incubation, with or
`without preimmune primary antibody, to samples incubated with the
`hMSH5 primary antibody. Testis tissues were obtained from surgical
`resections and contained evidence of testicular tumors. However, we
`confined our examination of hMSH5 expression to tissue regions that
`displayed clear evidence of full sperm maturation. IHC suggested
`
`Fig. 3. hMSH5 mRNA expression in human
`tissues. A, tissue expression. A 2.5–2.6-kb fragment
`can be detected on a very high level in testis tissue.
`It is also expressed in ovary, bone marrow, lymph
`node, trachea, and neural tissues, although at a
`significantly lower level. PBL, peripheral blood leu-
`kocytes. B, Western analysis of hMSH5 expressed
`in Sf9 insect cells.
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`Fig. 4. Immunohistochemical analysis of hMSH5 and hMSH2 protein expression in human testis and seminoma. A–D, human testis sections stained with an hMSH5 primary
`antibody. Round (RS) and elongated (ES) spermatids stain positive, while spermatogonia A (SA) and B (SB) as well as mature spermatozoa (SZ) are negative for hMSH5. Leydig cells
`(LC) in the stroma between the seminiferous tubules show a granular nonspecific reaction with the detection system due to their endogenous biotin (see negative control; H). The arrow
`in D marks a spermatogonium A undergoing mitosis (M), which stains negative for hMSH5. E, human testis stained for hMSH2 expression. All stages of spermatogenesis including
`early primary spermatocytes (PS) are positive except for most round spermatids (RS), the elongated spermatids (ES), and the mature spermatozoa (SZ). F, seminoma stained for hMSH2,
`where all tumor cells stain strongly, whereas the stroma is negative. G, seminoma stained for hMSH5 showing negativity in the tumor cells. H, negative control containing PBS instead
`of the hMSH5 primary antibody. Note the nonspecific granular cytoplasmic staining of the Leydig cell (LC). I, schematic representation of the stages of spermatogenesis. Examples
`of intratubular neoplasia (IN) and Sertoli cells (SC) are shown in B and E.
`
`However, there appears to be a significant interaction between GST-
`do not significantly interact with any of the IVTT-hMSH(y) (Fig. 5).
`hMSH5 with IVTT-hMSH4, which results in a distinct band that is
`Interaction was confirmed by densitometric quantitation, which sug-
`completely absent in the negative control (Fig. 5C). Different reaction
`gested at least a 10-fold difference in activity (Fig. 5).
`buffers (phosphate buffer and variations in the concentrations of
`IVTT-hMSH5 does not appear to interact with GST-hMSH2, GST-
`NaCl, Tween 20, and BSA) did not alter the background or the
`hMSH3, or GST-hMSH6 fusion proteins but shows a strong interac-
`interaction (data not shown). For comparison, the levels of IVTT
`tion with GST-hMSH4 (Fig. 5D). In the reverse experiment, GST-
`expression used in these experiments are shown in Fig. 5F. We have
`hMSH5 displayed no interaction above background when incubated
`found that the levels of GST-MSH(X) fusion protein are always in
`with IVTT-hMSH2, IVTT-hMSH3, and IVTT-hMSH6 (Fig. 5C).
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`hMSH5, A MEIOSIS-SPECIFIC HUMAN MutS HOMOLOGUE
`
`Fig. 5. hMSH5 protein interaction. A, interaction of IVTT-hMSH2 with GST-hMSH3, GST-hMSH4, and GST-hMSH5; GST moiety alone serves as a negative control, and
`GST-hMSH3 as a positive control. B, interaction of IVTT-hMSH3 with GST-hMSH2, GST-hMSH4, and GST-hMSH5; GST moiety alone serves as a negative control and GST-hMSH2
`as a positive control. C, interaction of IVTT-hMSH4 with GST-hMSH2, GST-hMSH3, GST-hMSH5, and GST-hMSH6; GST moiety alone serves as a negative control. D, interaction
`of IVTT-hMSH5 with GST-hMSH2, GST-hMSH3, GST-hMSH4, and GST-hMSH6; GST moiety alone serves as a negative control. E, interaction of IVTT-hMSH6 with GST-hMSH2,
`GST-hMSH4, and GST-hMSH5; GST moiety alone serves as a negative control and GST-hMSH2 as a positive control. F, expression analysis of IVTT-hMSH2, IVTT-hMSH3,
`IVTT-hMSH4, IVTT-hMSH5, and IVTT-hMSH6. Equal volumes (1 ml) of the IVTT extract were loaded. Intensities suggest the relative amounts of each IVTT protein introduced into
`the GST binding reactions. Right, positions of the molecular weight markers (in thousands).
`
`vast excess of the IVTT material, suggesting that specific interaction
`is related to the amount of IVTT material that associates with the
`GST-fusion protein (7). Taken together, these results suggest that
`hMSH5 specifically interacts with hMSH4 alone.
`
`Discussion
`
`We have identified and partially characterized the human homo-
`logue of the S. cerevisiae MSH5, hMSH5. In yeast, msh5 mutants
`have decreased spore viability, increased levels of meiosis I chromo-
`somal nondisjunction, and decreased levels of reciprocal exchange
`between, but not within, chromosomes (24). These results are con-
`sistent with a defect in meiotic processing. We have found that
`hMSH5 is located on chromosome 6p22-21 and is expressed at very
`high levels in the testis, where meiosis occurs continually throughout
`adult life. The cloning of hMSH5 has also been reported by another
`group (19). The chromosomal localization was determined to be on
`chromosome 6p21.3, and the tissue expression was described as
`ubiquitous, with the highest levels occurring in testis (19). We find
`hMSH5 to be expressed primarily in the testis, but it does not appear
`to be ubiquitously expressed. IHC on testicular sections revealed that
`hMSH5 was expressed in developing round and elongated spermatids.
`Spermatogonia and early primary spermatocytes were always com-
`pletely negative, and the expression of hMSH5 stops abruptly with the
`development of mature sperm. Although we can clearly identify the
`spermatids, the secondary spermatocytes are very hard to recognize
`because the transit between the primary spermatocyte (where meiosis
`I occurs) to the spermatid occurs rapidly. Because the expression of
`hMSH5 is exceedingly strong in the round spermatocytes, it is likely
`that the expression of hMSH5 originates in the late primary or the
`secondary spermatocyte, suggesting that hMSH5 expression is initi-
`ated in late meiosis I or meiosis II. The expression pattern of hMSH5
`would appear to be consistent with the phenotypes exhibited in yeast
`because the meiosis I chromosomal nondisjunction would occur at the
`cellular division between the primary and secondary spermatocyte (at
`just the stage where the expression of hMSH5 is likely to be initiated).
`We also observed low level expression of hMSH5 mRNA in a few
`other tissues. The most interesting are the bone marrow and lymph
`node, where T-cell and B-cell development takes place. At present, we
`have been unable to examine the expression of the hMSH5 protein in
`these tissues because normal tissues could not be obtained. However,
`we were still able to observe some full-length hMSH5 protein ex-
`pressed in a tonsil surgical sample (a repository of developing B
`cells). These results suggest that hMSH5 may play a role in both B-
`821
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`and perhaps T-cell development and that defects in hMSH5 might
`result in hematological defects.
`hMSH5 appears to specifically interact with hMSH4. No interac-
`tion with hMSH5 above background was observed for hMSH2,
`hMSH3, or hMSH6. Likewise, hMSH4 does not seem to interact with
`hMSH2, hMSH3, or hMSH6. Thus, it is likely that the hMSH4-
`hMSH5 heterodimer is specific and constitutes a functional interac-
`tion that is separate from hMSH2-hMSH3 and hMSH2-hMSH6 het-
`erodimers. Because this heterodimer constitutes the third identified
`interaction between human MutS homologues as well as the fact that
`this heterodimer appears to function very differently from the progen-
`itor bacterial MutS, the MutS (greek letter) nomenclature adopted by
`others would seem both inappropriate and nondescript (5). However,
`based on the conservation of the adenine nucleotide binding and
`hydrolysis domain, it is likely that the hMSH4-hMSH5 heterodimer
`also functions as a molecular switch (8). Although the control of the
`hMSH2-hMSH3 and hMSH2-hMSH6 molecular switches is mis-
`match provoked