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Expression of the Mutl Homologue Hmlh3 in Human Cells and Its Role in DNA Mismatch Repair

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Expression of the Mutl Homologue Hmlh3 in Human Cells and Its Role in DNA Mismatch Repair Research Article Expression of the MutL Homologue hMLH3 in Human Cells and its Role in DNA Mismatch Repair Elda Cannavo,1 Giancarlo Marra,1 Jacob Sabates-Bellver,1 Mirco Menigatti,2 Steven M. Lipkin,3 Franziska Fischer,1 Petr Cejka,1 and Josef Jiricny1 1Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland; 2Department of Internal Medicine, Faculty of Medicine, University of Modena, Modena, Italy; and 3Departments of Medicine and Biological Chemistry, University of California, Irvine, Irvine, California Abstract could be corroborated by analysis of the phenotypes of MMR- The human mismatch repair (MMR) proteins hMLH1 and deficient cells: Those lacking hMSH2 are fully MMR deficient and hPMS2 function in MMR as a heterodimer. Cells lacking either display a mutator phenotype and microsatellite instability that is protein have a strong mutator phenotype and display micro- consistent with the loss of repair of both base-base mismatches satellite instability, yet mutations in the hMLH1 gene account and insertion/deletion loops. Cells lacking hMSH6 retain a strong for f50% of hereditary nonpolyposis colon cancer families, mutator phenotype but their microsatellite instability is limited to mononucleotide repeats due to the functional redundancy with whereas hPMS2 mutations are substantially less frequent and h less penetrant. Similarly, in the mouse model, Mlh1À/À animals hMutS in insertion/deletion loop repair. This situation is mirrored are highly cancer prone and present with gastrointestinal in hereditary nonpolyposis colon cancer families, where the À/À penetrance of hMSH2 mutations is substantially higher than that tumors at an early age, whereas Pms2 mice succumb to cancer much later in life and do not present with gastro- of alterations in the hMSH6 locus (reviewed in ref. 2). a h intestinal tumors. This evidence suggested that MLH1 might Whereas it is generally accepted that hMutS and hMutS are functionally interact with another MutL homologue, which the mismatch recognition factors that initiate MMR (reviewed in compensates, at least in part, for a deficiency in PMS2. Sterility ref. 3), the function of the MutL homologues remains speculative. of Mlh1À/À, Pms2À/À, and Mlh3À/À mice implicated the Mlh1/ The human genome contains numerous genes that have significant Pms2 and Mlh1/Mlh3 heterodimers in meiotic recombination. sequence homology to mutL and to yeast MutL homologue and a We now show that the hMLH1/hMLH3 heterodimer, hMutL;, postmeiotic segregation genes; however, to date, only hMutL , can also assist in the repair of base-base mismatches and single a heterodimer of hMLH1 and hPMS2, could be shown to be extrahelical nucleotides in vitro. Analysis of hMLH3 expression involved in MMR. Correspondingly, hMLH1- or hPMS2-deficient in colon cancer cell lines indicated that the protein levels cells have a strong mutator phenotype and high microsatellite a vary substantially and independently of hMLH1. If hMLH3 instability (reviewed in ref. 1). In in vitro studies, hMutL could a participates in MMR in vivo, its partial redundancy with be shown to associate with hMutS on a mismatch-containing hPMS2, coupled with the fluctuating expression levels of substrate (4) and was suggested to act as a ‘‘molecular matchmaker’’ hMLH3, may help explain the low penetrance of hPMS2 between these protein complexes and the downstream effectors of h mutations in hereditary nonpolyposis colon cancer families. repair (reviewed in ref. 3). hMutL , a heterodimer of hMLH1 and (Cancer Res 2005; 65(23): 10759-66) hPMS1, has been biochemically characterized but could not be shown to participate in MMR in vitro (5). This finding was substantiated by in vivo evidence: Mice carrying a disruption in Introduction the Pms1 gene display neither microsatellite instability nor cancer Mismatch repair (MMR) proteins are a highly conserved group of predisposition (6). hMLH3 was identified through its interaction polypeptides that play key roles in the correction of mispairs with hMLH1 on Far Western blots (7); however, this heterodimer, arising during DNA replication. They also prevent recombination hMutLg, has not been biochemically characterized and its role between nonidentical sequences and participate in the signaling of in mammalian MMR has not been established. MLH3 was first certain types of DNA damage. The importance of MMR proteins in identified in Saccharomyces cerevisiae and its gene product, the maintenance of genomic integrity is underscored by the finding scMlh3p, was shown to bind scMlh1p (8, 9) and to be involved in that germ line mutations in MMR genes predispose to hereditary meiotic recombination (reviewed in refs. 10, 11). As mlh3 mutants nonpolyposis colon cancer, a common familial cancer predisposi- display a mutator phenotype similar to that of msh3-deficient strains tion syndrome (reviewed in refs. 1, 2). The principal MMR players (8, 12), it was suggested that the two polypeptides are involved in the in human cells are homologues of the bacterial MutS and MutL repair of a subset of insertion/deletion loops. hMLH3 seems to be proteins. hMutSa, a heterodimer of the MutS homologues hMSH2 involved in meiotic recombination (13, 14) and the same is true for and hMSH6, binds base-base mismatches and small insertion/ the murine Mlh3 (14). As both Mlh1- and Mlh3-deficient mice are deletion loops, whereas hMutSh (a heterodimer of hMSH2 and sterile (reviewed in refs. 10, 11), it was suggested that the two hMSH3) binds only insertion/deletion loops. This in vitro evidence polypeptides function together. However, unlike Mlh1À/À animals (6), Mlh3À/À mice did not succumb to cancer in the first 9 months of life (15). The roles of the various MMR factors and the pheno- Requests for reprints: Josef Jiricny, Institute of Molecular Cancer Research, types of mice with defects in MMR genes are listed in Table 1. University of Zu¨rich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Phone: The involvement of MutL homologue malfunctions in human 411-634-8910; Fax: 411-634-8903; E-mail: [email protected]. I2005 American Association for Cancer Research. cancer is not as clear cut as in the case of the MutS homologues. doi:10.1158/0008-5472.CAN-05-2528 Mutations in hMLH1 predominate in hereditary nonpolyposis www.aacrjournals.org 10759 Cancer Res 2005; 65: (23). December 1, 2005 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research Table 1. Overview of mammalian MutS and MutL homologues and their roles in MMR Heterodimer Components MMR role Phenotype of knockout mice hMutSa hMSH2 Repair of base-base mismatches and small loops Lymphomas, gastrointestinal, skin, and other tumors hMSH6 Lymphomas, gastrointestinal, and other tumors hMutSh hMSH2 Repair of loops Lymphomas, gastrointestinal, skin, and other tumors hMSH3 Gastrointestinal tumors hMutLa hMLH1 Repair of all MMR substrates Lymphomas, gastrointestinal, skin and other tumors; sterility hPMS2 Lymphomas, sarcomas; male sterility hMutLh hMLH1 ? Lymphomas, gastrointestinal, skin, and other tumors hPMS1 No phenotype hMutLg hMLH1 ? Lymphomas, gastrointestinal, skin, and other tumors hMLH3 Sterility colon cancer, accounting for nearly 50% of all known germ line Expression of hMutL; MMR gene mutations (2). Surprisingly, no germ line mutations The Bac-to-Bac baculovirus expression system (Life Technologies, have been found in hPMS1 or hPMS2, which was unexpected, given Gaithersburg, MD) was used according to the instructions of the manu- Â 8 the key role of the latter protein in MMR. Recent immunohisto- facturer. Spodoptera frugiperda Sf9 cells (2 10 ; Life Technologies) were infected with either a single recombinant baculovirus or with a combina- chemical analysis of 1,048 unselected colon tumors revealed the f tion of two viruses at a multiplicity of infection of 10. Cells were harvested lack of hPMS2 in 1.5%, a proportion similar to that of MSH2- 72 hours after infection and total extracts were prepared as described (21). deficient cancers (16). Genetic analysis identified germ line Partial purification of hMutLg from Sf9 extracts was done using Ni-NTA mutations in hPMS2 in a number of these patients and it is likely agarose (Qiagen, Hilden, Germany), and the QIAexpressionist system was that the remainder will also be linked to genetic alterations once used according to the instructions of the manufacturer using 5 mL of 50% the problems associated with sequencing of the hPMS2 locus are Ni-NTA slurry per 100 mg of protein extract. overcome (there are f20 hPMS2 pseudogenes on chromosome 7, hMutLg was expressed also in bacteria using a bicistronic vector which interfere with DNA sequencing). However, these patients do pET11b-His6-hMLH3/MLH1 (cloning information on request) in the BL21 j not belong to typical hereditary nonpolyposis colon cancer families strain of Escherichia coli. After induction of expression at 37 C for 4 hours with 0.4 mmol/L isopropyl-h-D-thiogalactopyranoside, the heterodimer was and the penetrance of these mutations seems to be very low. One expressed but was insoluble. Nevertheless, the protein could be used to possible explanation for this finding is that the defect in hPMS2 is quantify the relative abundance of hMLH3 in HeLa cells. partially compensated for by another MutL homologue, such as hMLH3. Germ line hMLH3 missense and frameshift mutations hMLH3 Antibody Production and Purification have been described in familial colorectal cancer cases but the The COOH-terminal polypeptide of hMLH3 (amino acids 961-1,453) was implication of these alterations in carcinogenesis is ambiguous. In expressed using the Impact-CN-System (New England Biolabs) in BL21 E. coli transformed with pTXB1-hMLH3 (amino acids 961-1,453). The some cases, the mutation in hMLH3 was identified in families peptide was purified using fast protein liquid chromatography on a MiniQ carrying a second MMR gene mutation, whereas no mutations in 4.6/50 PE column (Amersham Pharmacia, Uppsala, Sweden) and used to the other MMR genes could be identified in other cases (17–19). A immunize rabbits at Eurogentec (Seraing, Belgium).
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