The DNA mismatch repair system (MMR) is essential for maintaining the integrity of the mammalian genome. Mutations in MMR genes can lead to increased cancer susceptibility and meiotic failure. At present most of our understanding of the MMR is limited to the early steps of MMR. The initial events include the recognition of mispaired nucleotides by the mammalian homologs of the E. coli mutS gene. Subsequent to the mismatch recognition event, the MutS proteins bind to MutL proteins. This interaction activates events that lead to the excision of the misincorporated nucleotide(s) and the filling of the resulting single strand gap by DNA resynthesis. These later MMR events are less well understood and many of the enzymes facilitating these reactions are unknown. An essential step for late MMR is the removal of the newly synthesized DNA strand by exonucleases. Data in S. cerevisiae indicate that 5'-3' as well as 3'-5' exonucleases are involved in this process. Recently, yeast Exonuclease l (EXO1), a 5'-3' exonuclease, has been identified due to its interaction with MSH2 in two hybrid screens. EXO1 deficient yeast strains display a mutator phenotype that is similar to MSH2 deficient strains, providing strong evidence for a role in MMR. In addition to its role in MMR, EXO1 is also required for mitotic and meiotic recombination in yeast. Based on the studies in yeast, we hypothesize that EXO1 is an essential component of mammalian MMR and targeted disruption of the Exonuclease 1 gene in mice will result in MMR deficiency leading to cancer and infertility.
The specific aims of this proposal are: 1. To investigate the role of EXO1 in mammalian Meiosis: We have generated a mouse line carrying an inactivating mutation. Homozygous mutant Exo1 mice are infertile and we will analyze meiosis in the Exo1 mutant mice. We propose to identify the meiotic MMR partners of EXO1 and analyze their interactions. 2. To examine the role of mammalian EX01 in mitotic MMR and recombination: We will provide a detailed study of the role of EX01 in MMR and recombination in EXO1 deficient embryonic stem cell lines. 3. To study the cancer susceptibility of EXO1 deficient mice: We will analyze the cancer susceptibility phenotype of Exo1 mutant mice to determine the involvement of EX01 in the initiation and progression of cancer. 4. To determine the relationship between EXO1 and other MMR genes: In order to position EXO1 in the MMR pathway, we will examine the repair activity of the MSH2-MSH6 and MSH2-MSH3 complexes on a mutant Exo1 background to determine whether it is necessary and sufficient for the activity of either or both of these complexes.
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