R. Michael Liskay

Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over.

Mice that are deficient in either the Pms2 or Msh2 DNA mismatch repair genes have microsatellite instability and a predisposition to tumours. Interestingly, Pms2–deficient males display sterility associated with abnormal chromosome pairing in meiosis. Here mice deficient in another mismatch repair gene, Mlh1, possess not only microsatellite instability but are also infertile (both males and females). Mlh 1 –deficient spermatocytes exhibit high levels of prematurely separated chromosomes and arrest in first division meiosis. We also show that Mlh1 appears to localize to sites of crossing over on meiotic chromosomes/Together these findings suggest that Mlh1 is involved in DNA mismatch repair and meiotic crossing over.

Requirement for PCNA in DNA Mismatch Repair at a Step Preceding DNA Resynthesis

A two-hybrid system was used to screen yeast and human expression libraries for proteins that interact with mismatch repair proteins. PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2. A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1. Mismatch repair activity was examined in human cell extracts using an assay that does not require DNA repair synthesis. Activity was inhibited by p21WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA. The data suggest a PCNA requirement in mismatch repair at a step preceding DNA resynthesis. The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and replication and may be relevant to the roles of mismatch repair proteins in other DNA transactions.

Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis

Using gene targeting in embryonic stem cells, we have derived mice with a null mutation in a DNA mismatch repair gene homolog, PMS2. We observed microsatellite instability in the male germline, in tail, and in tumor DNA of PMS2-deficient animals. We therefore conclude that PMS2 is involved in DNA mismatch repair in a variety of tissues. PMS2-deficient animals appear prone to sarcomas and lymphomas. PMS2-deficient males are infertile, producing only abnormal spermatozoa. Analysis of axial element and synaptonemal complex formation during prophase of meiosis I indicates abnormalities in chromosome synapsis. These observations suggest links among mismatch repair, genetic recombination, and chromosome synapsis in meiosis.

Differences in the DNA of the inactive X chromosomes of fetal and extraembryonic tissues of mice

We have examined the role of DNA modification in X chromosome inactivation of fetal tissues of the mouse using DNA-mediated gene transfer for the gene hypoxanthine phosphoribosyltransferase (HPRT). Two types of tissues have been examined with respect to randomness of inactivation in 14-day mouse conceptuses: 1) fetal tissue, which undergoes random inactivation of either the maternal or paternal X; and 2) yolk sac endoderm tissue, an extraembryonic membrane, which normally undergoes nonrandom inactivation of the paternal X. Exploiting an electrophoretic variant of HPRT as a means to mark the active and inactive HPRT alleles we provide evidence that: 1) inactive X DNA of the fetus at 14 days behaves like that of both adult tissue and cell lines in that the inactive X DNA is not efficient in gene transfer; and 2) in contrast, inactive X DNA from yolk sac endoderm is functional in gene transfer. Thus, despite the similarity in single active X chromosome expression in yolk sac endoderm and somatic tissues, there appears to be a difference at the level of DNA modification between these two tissues.

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2–MSH3 or MSH2–MSH6) or crossing over (MSH4–MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4–MSH5. The second complex involves MLH3 together with MSH2–MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2 − / − males, but not females, providing an explanation for the sexual dimorphism seen in Pms2 − / − mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2–MSH3 and is decreased in Msh2 − / − and Msh3 − / − mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

Contributions by MutL Homologues Mlh3 and Pms2 to DNA Mismatch Repair and Tumor Suppression in the Mouse

Germ line DNA mismatch repair mutations in MLH1 and MSH2 underlie the vast majority of hereditary non-polyposis colon cancer. Four mammalian homologues of Escherichia coli MutL heterodimerize to form three distinct complexes: MLH1/PMS2, MLH1/MLH3, and MLH1/PMS1. Although MLH1/PMS2 is generally thought to have the major MutL activity, the precise contributions of each MutL heterodimer to mismatch repair functions are poorly understood. Here, we show that Mlh3 contributes to mechanisms of tumor suppression in the mouse. Mlh3 deficiency alone causes microsatellite instability, impaired DNA-damage response, and increased gastrointestinal tumor susceptibility. Furthermore, Mlh3;Pms2 double-deficient mice have tumor susceptibility, shorter life span, microsatellite instability, and DNA-damage response phenotypes that are indistinguishable from Mlh1-deficient mice. Our data support previous results from budding yeast that show partial functional redundancy between MLH3 and PMS2 orthologues for mutation avoidance and show a role for Mlh3 in gastrointestinal and extragastrointestinal tumor suppression. The data also suggest a mechanistic basis for the more severe mismatch repair-related phenotypes and cancer susceptibility in Mlh1- versus Mlh3- or Pms2-deficient mice. Contributions by both MLH1/MLH3 and MLH1/PMS2 complexes to mechanisms of mismatch repair-mediated tumor suppression, therefore, provide an explanation why, among MutL homologues, only germ line mutations in MLH1 are common in hereditary non-polyposis colon cancer.

Evidence for Intrachromosomal Gene Conversion in Cultured Mouse Cells

In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK- cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK+ was applied to detect recombinational events between different TK- genes. TK+ segregants were observed at a frequency of 10(-4)-10(-5) in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK+ cells at frequencies of 10(-7) or less. Physical analysis of the TK+ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho l endonuclease digestion in each TK+ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.

Mlh1 deficiency enhances several phenotypes of Apc(Min)/+ mice.

Defects in APC and DNA mismatch repair genes are associated with a strong predisposition to colon cancer in humans, and numerous mouse strains with mutations in these genes have been generated. In this report we describe the phenotype of Min/+ Mlh1−/− mice. We find that these doubly mutant mice develop more than three times the number of intestinal adenomas compared to Min/+ Mlh1+/+ or +/− mice but that these tumors do not show advanced progression in terms of tumor size or histological appearance. Full length Apc protein was not detected in the tumor cells from Min/+ Mlh1−/− mice. Molecular analyses indicated that in many tumors from Min/+ Mlh1−/− mice, Apc was inactivated by intragenic mutation. Mlh1 deficiency in Min/+ mice also led to an increase in cystic intestinal crypt multiplicity as well as enhancing desmoid tumorigenesis and epidermoid cyst development. Thus, Mlh1 deficiency influences the somatic events involved in the development of most of the phenotypes associated with the Min mutation.

Cell-cycle characteristics of undifferentiated and differentiating embryonal carcinoma cells

Abstract The cell-cycle parameters of undifferentiated and differentiating embryonal carcinoma cells were determined. Undifferentiated cultures of an F9 subclone, OTF9-63, had a 12-hr generation time, 10-hr S, and 2-hr G2 + M. G1 was less than 0.5 hr. In contrast, OTF9-63 cells induced to differentiate by treatment with retinoic acid had a 16.8-hr generation time, 12.5-hr S, 2-hr G2 + M, and 2.3-hr G1. Similar results were obtained with undifferentiated cultures and aggregation-induced differentiating cultures of PSA-1 cells. These data demonstrate that the undifferentiated stem cells have little or no G1, and that both G1 and S lengthen during differentiation.

Elevated mutant frequencies and increased C : G-->T : A transitions in Mlh1-/- versus Pms2-/- murine small intestinal epithelial cells.

Mutations in DNA mismatch repair (MMR) genes are associated with increased genomic instability and susceptibility to cancer. Mice rendered deficient in either Mlh1 or Pms2 as a result of gene targeting are prone to tumorigenesis, particularly, lymphomas. In addition, although Mlh1−/− mice also develop small intestinal adenomas and adenocarcinomas, Pms2−/− animals remain free of such tumors. To establish whether this phenotypic dichotomy might be associated with a quantitative and/or qualitative difference in genomic instability in these mice, we determined small intestinal epithelial cell DNA mutant frequency and mutation spectrum using a transgenic λ-phage lacI reporter system. Mutant frequencies obtained from both Mlh1−/− and Pms2−/− mice revealed elevations of 18- and 13-fold, respectively, as compared to their wild-type littermates. Interestingly, we found that C : G→T : A transitions were significantly elevated in Mlh1−/− mice, accounting in large measure for the 1.5-fold lacI mutant frequency increase seen in these animals. We hypothesize that the increased level of C : G→T : A mutations may explain, in part, why Mlh1−/− mice, but not Pms2−/− mice, develop small intestinal tumors. Furthermore, the difference in the lacI mutational spectrum of Mlh1−/− and Pms2−/− mice suggests that other MutL-like heterodimers may play important roles in the repair of G : T mispairs arising within murine small intestinal epithelial cells.