This proposal focuses on several mutational changes accompanying DNA repair in budding yeast. DNA repair is induced by galactose-regulated expression of the site-specific HO endonuclease, creating a single double-strand break (DSB). One major goal is to understand complex mutations associated by template switching that occur during gene conversion. Two types of repair will be studied: 1. Quasipalindrome mutation formation during gene conversion and 2. Interchromosomal microhomology-mediated template switching during gene conversion. Genetic analysis of helicases and other repair factors will be screened to find proteins that control the level of these two events, along with an exploration of the role of chromatin. A second goal is to understand changes in repeat copy number during gene conversion, motivated by our recent discovery of important differences between DSB break repair and gap repair. In collaboration with Mitch McVey, another member of this Program Project who focuses on DSB repair in fruit flies, we will assess the frequency of abortive gap repair leading to deletions between repeated sequences within the copied region.
Mutations associated with repair of chromosomal breaks are an important factor in the origin of human disease. Our recent work has also shed new light on the origin of quasipalindrome mutations. Most surprising was our finding that three types of mutations characterized as template switches (frameshifts, quasipalindromes and interchromosomal homeologous recombination events) all were driven by the wild type DNA polymerase 5. Further characterization of these types of events and the proteins that normally prevent their appearance is a fundamentally important goal in understanding the origin of mutations affecting human health and disease.
|Dwivedi, Gajendrahar; Haber, James E (2018) Assaying Mutations Associated With Gene Conversion Repair of a Double-Strand Break. Methods Enzymol 601:145-160|
|Kononenko, Artem V; Ebersole, Thomas; Vasquez, Karen M et al. (2018) Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system. Nat Struct Mol Biol 25:669-676|
|McGinty, Ryan J; Mirkin, Sergei M (2018) Cis- and Trans-Modifiers of Repeat Expansions: Blending Model Systems with Human Genetics. Trends Genet 34:448-465|
|Polleys, Erica J; Freudenreich, Catherine H (2018) Methods to Study Repeat Fragility and Instability in Saccharomyces cerevisiae. Methods Mol Biol 1672:403-419|
|Gallagher, Danielle N; Haber, James E (2018) Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing. ACS Chem Biol 13:397-405|
|Radchenko, Elina A; McGinty, Ryan J; Aksenova, Anna Y et al. (2018) Quantitative Analysis of the Rates for Repeat-Mediated Genome Instability in a Yeast Experimental System. Methods Mol Biol 1672:421-438|
|Moore, Anthony; Dominska, Margaret; Greenwell, Patricia et al. (2018) Genetic Control of Genomic Alterations Induced in Yeast by Interstitial Telomeric Sequences. Genetics 209:425-438|
|Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun et al. (2018) CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles. Proc Natl Acad Sci U S A 115:E2040-E2047|
|Haber, James E (2018) DNA Repair: The Search for Homology. Bioessays 40:e1700229|
|Polleys, Erica J; House, Nealia C M; Freudenreich, Catherine H (2017) Role of recombination and replication fork restart in repeat instability. DNA Repair (Amst) 56:156-165|
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