The main goal of our research is to analyze the biological functions of the mammalian DNA mismatch repair system (MMR) and to determine how mutations in MMR genes affect DNA repair and cancer susceptibility. MMR is essential for maintaining the integrity of the mammalian genome and mutations in MMR genes result in increased cancer susceptibility and meiotic failure. Eukaryotic MMR is a complex system that requires the interaction of several MutS and MutL proteins for the initiation of the repair reaction. Subsequent to mismatch recognition, downstream events are activated that lead to the excision of the misincorporated nucleotide(s) and the filling in of the resulting single strand gap by DNA resynthesis. In the past funding period, we performed a comprehensive analysis of Exonuclease 1 (Exol) mutant mice, the only currently identified exonuclease known to function in the eukaryotic excision reaction. We determined that Exol functions in the Msh2-Msh6-dependent repair of base-base mismatches and that Exol inactivation causes a highly penetrant cancer predisposition phenotype. In addition, loss of Exol function caused infertility in male and female mice, indicating an essential role for Exol in mammalian meiosis. We hypothesize that the generation of a set of targeted missense mutations will elucidate how Exol functions to suppress cancer and will allow a study of its role in meiosis. In addition, efforts to identify proteins that interact with Exol, will help elucidate other key components of the late stages of MMR.
The specific aims of this proposal are: 1. To model human Exol missense mutations found in human cancer patients as well as prevalent coding single nucleotide polymorphisms (SNPs) in mice and analyze the resulting cancer susceptibility phenotype. 2. To determine the effect of Exol missense mutations on MMR and mutation avoidance. We will analyze the effect of the Exol knock-in mutations on DNA repair functions in vitro and determine the resulting in vivo mutator phenotype in mouse tissues. 3. To determine the effects of missense mutations on the biological functions of Exol in mammalian meiosis. We will perform detailed histopathological and cytogenetic studies to determine the mechanisms by which Exol missense mutations may disrupt prophase I progression in spermatocytes and oocytes. 4. To analyze the significance of a novel Exol interacting protein for MMR and to establish a system for the in vivo analysis of MMR complexes. We have identified RuvBL2 helicase as an Exol interacting enzyme and will study its potential role in MMR. We also propose to establish an in vivo system for the analysis of MMR complex formation and the identification of novel MMR associated proteins in mouse tissue.
|Chen, Chun-Chin; Avdievich, Elena; Zhang, Yongwei et al. (2017) EXO1 suppresses double-strand break induced homologous recombination between diverged sequences in mammalian cells. DNA Repair (Amst) 57:98-106|
|Edwards, Yasmin (2017) An Msh3 ATPase domain mutation has no effect on MMR function. BMC Res Notes 10:616|
|Rein, Katrin; Yanez, Diana A; Terré, Berta et al. (2015) EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele. Nucleic Acids Res 43:7371-87|
|Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried (2014) Seamless Ligation Cloning Extract (SLiCE) cloning method. Methods Mol Biol 1116:235-44|
|van Oers, J M M; Edwards, Y; Chahwan, R et al. (2014) The MutS? complex is a modulator of p53-driven tumorigenesis through its functions in both DNA double-strand break repair and mismatch repair. Oncogene 33:3939-46|
|Dietlein, Felix; Thelen, Lisa; Jokic, Mladen et al. (2014) A functional cancer genomics screen identifies a druggable synthetic lethal interaction between MSH3 and PRKDC. Cancer Discov 4:592-605|
|Schaetzlein, Sonja; Chahwan, Richard; Avdievich, Elena et al. (2013) Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes. Proc Natl Acad Sci U S A 110:E2470-9|
|Bothmer, Anne; Rommel, Philipp C; Gazumyan, Anna et al. (2013) Mechanism of DNA resection during intrachromosomal recombination and immunoglobulin class switching. J Exp Med 210:115-23|
|Ene, Adriana C; Park, Stephanie; Edelmann, Winfried et al. (2013) Caspase 9 is constitutively activated in mouse oocytes and plays a key role in oocyte elimination during meiotic prophase progression. Dev Biol 377:213-23|
|Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40:e55|
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