Biomedical subjects

R Michael Liskay

Publications and source records attributed to R Michael Liskay.

15 recordsLinked to original sources

PubMed2006

The E705K mutation in hPMS2 exerts recessive, not dominant, effects on mismatch repair.

The hPMS2 mutation E705K is associated with Turcot syndrome. To elucidate the pathogenesis of hPMS2-E705K, we modeled this mutation in yeast and characterized its expression and effects on mutation avoidance in mammalian cells. We found that while hPMS2-E705K (pms1-E738K in yeast) did not significantly affect hPMS2 (Pms1p in yeast) stability or interaction with MLH1, it could not complement the mutator phenotype in MMR-deficient mouse or yeast cells. Furthermore, hPMS2-E705K/pms1-E738K inhibited MMR in wild-type (WT) mammalian cell extracts or yeast cells only when present in excess amounts relative to WT PMS2. Our results strongly suggest that hPMS2-E705K is a recessive loss-of-function allele.

Adenosine Triphosphatases↗

PubMed2006

Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6.

Defects in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer. Because the MMR pathway includes multiple factors with both overlapping and divergent functions, we sought to compare the impact of deficiencies in each of several MMR genes on genetic instability using a collection of knock-out mouse models. We investigated mutation frequencies and patterns in MMR-deficient mice using two transgenic reporter genes, supFG1 and cII, in the context of mice deficient for Pms2, Mlh1, Msh2, Msh3 or Msh6 or both Msh2 and Msh3 or both Msh3 and Msh6. We found that the mean mutation frequencies of all of the MMR-deficient mice were significantly higher than the mean mutation frequencies of wild-type mice. Mlh1-deficient mice and Msh2-deficient mice had the highest mutation frequencies in a comparison of the single nullizygous mice. Of all the mice studied, mice nullizygous for both Msh2 and Msh3 and those nullizygous for both Msh3 and Msh6 displayed the greatest overall increases in mutation frequencies compared with wild-type mice. Sequence analysis of the mutated reporter genes revealed significant differences between the individual groups of MMR-deficient mice. Taken together, our results further characterize the functions of the MMR factors in mutation avoidance and provide in vivo correlation to biochemical models of the MMR pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance.

Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.

Adenosine Triphosphatases↗

PubMed2005

Crossover and noncrossover pathways in mouse meiosis.

During meiosis, recombination between homologous chromosomes generates crossover (CR) and noncrossover (NCR) products. CRs establish connections between homologs, whereas intermediates leading to NCRs have been proposed to participate in homologous pairing. How these events are differentiated and regulated remains to be determined. We have developed a strategy to detect, quantify, and map NCRs in parallel to CRs, at the Psmb9 meiotic recombination hot spot, in male and female mouse germ lines. Our results report direct molecular evidence for distinct CR and NCR pathways of DNA double-strand break (DSB) repair in mouse meiosis based on three observations: both CRs and NCRs require Spo11, NCR products have shorter conversion tracts than CRs, and only CRs require the MutL homolog Mlh1. We show that both products are formed from middle to late pachytene of meiotic prophase and provide evidence for an Mlh1-independent CR pathway, where mismatch repair does not require Mlh1.

Adaptor Proteins, Signal Transducing↗

PubMed2005

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.

Adaptor Proteins, Signal Transducing↗

PubMed2005

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.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Novel PMS1 alleles preferentially affect the repair of primer strand loops during DNA replication.

Null mutations in DNA mismatch repair (MMR) genes elevate both base substitutions and insertions/deletions in simple sequence repeats. Data suggest that during replication of simple repeat sequences, polymerase slippage can generate single-strand loops on either the primer or template strand that are subsequently processed by the MMR machinery to prevent insertions and deletions, respectively. In the budding yeast Saccharomyces cerevisiae and mammalian cells, MMR appears to be more efficient at repairing mispairs comprised of loops on the template strand compared to loops on the primer strand. We identified two novel yeast pms1 alleles, pms1-G882E and pms1-H888R, which confer a strong defect in the repair of "primer strand" loops, while maintaining efficient repair of "template strand" loops. Furthermore, these alleles appear to affect equally the repair of 1-nucleotide primer strand loops during both leading- and lagging-strand replication. Interestingly, both pms1 mutants are proficient in the repair of 1-nucleotide loop mispairs in heteroduplex DNA generated during meiotic recombination. Our results suggest that the inherent inefficiency of primer strand loop repair is not simply a mismatch recognition problem but also involves Pms1 and other proteins that are presumed to function downstream of mismatch recognition, such as Mlh1. In addition, the findings reinforce the current view that during mutation avoidance, MMR is associated with the replication apparatus.

Adaptor Proteins, Signal Transducing↗

PubMed2004

EXO1-A multi-tasking eukaryotic nuclease.

Exo1 was first isolated as a 5' --> 3' exonuclease activity induced during meiosis in fission yeast and since that time has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. Involvement in multiple pathways affecting genomic stability makes EXO1 a logical target for mutation during oncogenesis. Here, we review studies in several experimental systems that shed light on the role of Exo1 in these DNA transaction pathways, particularly those that may relate to oncogenesis.

Amino Acid Sequence↗

PubMed2003

Apoptosis and mutation in the murine small intestine: loss of Mlh1- and Pms2-dependent apoptosis leads to increased mutation in vivo.

The mismatch repair (MMR) protein Msh2 has been shown to function in the apoptotic response to alkylating agents in vivo. Here, we extend these studies to the MutL homologues (MLH) Mlh1 and Pms2 by analysing the apoptotic response within the small intestine of gene targeted strains. We demonstrate significant differences between Msh2, Mlh1 and Pms2 mutations in influencing apoptotic signalling following 50mg/kg N-methyl-nitrosourea (NMNU), with no obvious reliance upon either Mlh1 or Pms2. However, following exposure to 100mg/kg temozolomide or lower levels of NMNU (10mg/kg) both Mlh1- and Pms2-dependent apoptosis was observed, indicating that the apoptotic response at these levels of DNA damage is dependent on the MutL homologues. Given our ability to observe a MutLalpha dependence of the apoptotic response, we tested whether perturbations of this response directly translate into increases in mutation frequency in vivo. We show that treatment with temozolomide or 10mg/kg NMNU significantly increases mutation in both the Mlh1 and Pms2 mutant mice. At higher levels of NMNU, where the apoptotic response is independent of Mlh1 and Pms2, no gene dependent increase in mutation frequency was observed. These results argue that the MutSalpha and MutLalpha are not equally important in their ability to signal apoptosis. However, when MMR does mediate apoptosis, perturbation of this response leads to long-term persistence of mutant cells in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type.

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae.

Exo1p is a member of the Rad2p family of structure-specific nucleases that contain conserved N and I nuclease domains. Exo1p has been implicated in numerous DNA metabolic processes, such as recombination, double-strand break repair and DNA mismatch repair (MMR). In this report, we describe in vitro and in vivo characterization of full-length wild-type and mutant forms of Exo1p. Herein, we demonstrate that full-length yeast Exo1p possesses an intrinsic 5'-3' exonuclease activity as reported previously, but also possesses a flap-endonuclease activity. Our study indicates that Exo1p shares similar, but not identical structure-function relationships to other characterized members of the Rad2p family in the N and I nuclease domains. The two exo1p mutants we examined, showed deficiencies for both double-stranded DNA (dsDNA) 5'-3' exonuclease and flap-endonuclease activities. Examining the genetic interaction of these two exo1 mutations with rad27Delta suggest that the Exo1p flap-endonuclease activity and not the dsDNA 5'-3' exonuclease is redundant to Rad27p for viability. In addition, our in vivo results also indicate that many exo1Delta phenotypes are dependent on the complete catalytic activities of Exo1p. Finally, our findings plus those of other investigators suggest that Exo1p functions both in a catalytic and a structural capacity during DNA MMR.

Baculoviridae↗

PubMed2002

Contribution of human mlh1 and pms2 ATPase activities to DNA mismatch repair.

MutLalpha, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLalpha and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLalpha proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLalpha demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLalpha activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Diet, cancer and aging in DNA mismatch repair deficient mice.

Diet is an important risk factor for many cancers. High fat/low calcium (HFLC) diets are associated with increased tumorigenesis, whereas caloric restriction (CR) reproducibly increases lifespan and decreases tumors. Mutations are involved in aging and cancer, and different diets may alter mutagenesis. However, a number of repair pathways normally counteract mutations by correcting errors before they can be fixed in the genome. To further understand interactions between diet, aging and cancer, mice deficient in a major repair pathway called DNA mismatch repair (MMR) were fed HFLC, CR or control diets. Mlh1 deficient mice are prone to lymphomas and intestinal adenomas and carcinomas. No significant changes in adenocarcinoma or lymphoma incidence were observed with HFLC or CR diets. Significantly more (2.2-fold) adenomas occurred with HFLC diets although adenoma numbers were unchanged with CR. Only a small increase in lifespan (116% of control) was achieved with CR. In addition, levels of microsatellite mutations in the small and large intestines were unchanged with the different diets. Our studies indicate that MMR deficiency may be epistatic to certain otherwise strong environmental influences on carcinogenesis or aging.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Maternal effect for DNA mismatch repair in the mouse.

DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of approximately 10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F(1) progeny from Pms2(-/-) female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was approximately 9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.

Adenosine Triphosphatases↗

PubMed2002

Alleles of the yeast Pms1 mismatch-repair gene that differentially affect recombination- and replication-related processes.

Mismatch-repair (MMR) systems promote eukaryotic genome stability by removing errors introduced during DNA replication and by inhibiting recombination between nonidentical sequences (spellchecker and antirecombination activities, respectively). Following a common mismatch-recognition step effected by MutS-homologous Msh proteins, homologs of the bacterial MutL ATPase (predominantly the Mlh1p-Pms1p heterodimer in yeast) couple mismatch recognition to the appropriate downstream processing steps. To examine whether the processing steps in the spellchecker and antirecombination pathways might differ, we mutagenized the yeast PMS1 gene and screened for mitotic separation-of-function alleles. Two alleles affecting only the antirecombination function of Pms1p were identified, one of which changed an amino acid within the highly conserved ATPase domain. To more specifically address the role of ATP binding/hydrolysis in MMR-related processes, we examined mutations known to compromise the ATPase activity of Pms1p or Mlh1p with respect to the mitotic spellchecker and antirecombination activities and with respect to the repair of mismatches present in meiotic recombination intermediates. The results of these analyses confirm a differential requirement for the Pms1p ATPase activity in replication vs. recombination processes, while demonstrating that the Mlh1p ATPase activity is important for all examined MMR-related functions.

Adaptor Proteins, Signal Transducing↗