Repair of chromosome double-strand breaks (DSBs) is essential for viability in human cells and aberrant repair to genomic instability. This proposal continues the study of DSB repair by the most common pathway - gene conversion - in the model system, the budding yeast Saccharomyces cerevisiae. A detailed analysis of DSB repair is made possible by rapidly inducing a single DSB in all cells of the population, using a galactose-inducible HO endonuclease to effect mating-type (MAT) gene switching. Physical analysis of DNA solated from cells undergoing recombination, by southern blot and PCR analysis, makes it possible to identify intermediates of recombination, and chromatin immunoprecipitation (ChIP) permits one to flow the recruitment of recombination proteins in both wild type and cells lacking various recombination and DNA replication proteins. It is proposed to continue our analysis of early steps in homologous recombination, ncluding the characterization of strand invasion intermediates and the defects of mutant recombination proteins. The dynamics of homology searching will be investigated, using microscopic analysis of fluorescently-tagged segments adjacent to chromosomal regions undergoing recombination, as well as by Chip. Special attention will be given to the role of the cis-acting Recombination Enhancer. A detailed analysis of new DNA synthesis during gene conversion will be performed, including a study of recruitment of DNA polymerases to the strand invasion intermediate. Another finding that will be pursued concerns a 1000-fold increase in the rate of mutagenesis by template switching during gene conversion. The role of DNA processivity factors and helicases will be examined to identify the basis of template switching. In addition, the analysis of changes in chromatin structure accompanying MST switching will be pursued, using a modified MAT locus that allows the analysis of chromatin alterations and reestablishment during repair and the analysis of chromatin chaperones and remodelers. A second major theme of this proposal is to understand the role of the Recombination Enhancer (RE) in regulating the mating-type dependent choice of one of two alternative donors during MAT switching. The role of the phospho-threonine binding HA domain of Fkhl, which has been shown to be essential for donor preference when bound at RE, will be investigated. Identification of the phospho-threonine target in the region around the DSB will be carried out, both by characterization of the FHA domain and by analysis of DSB-dependent proteins that associate with the FHA domain.

Public Health Relevance

Human cells require homologous recombination by gene conversion in every cell cycle to repair the doublestrand breaks that arise during replication. Failure to repair these lesions leads to a high rate of genome instability and diseases including cancer. Our recent study of the elevated rate of mutations associated with gene conversion has revealed mechanisms of quasipalindrome formation and copy number variation that are also signatures of many cancers and other human diseases. These events can be studied in great detail using the budding yeast model system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37GM020056-40
Application #
8240171
Study Section
Special Emphasis Panel (NSS)
Program Officer
Janes, Daniel E
Project Start
1976-01-01
Project End
2017-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
40
Fiscal Year
2012
Total Cost
$557,860
Indirect Cost
$208,333
Name
Brandeis University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
Roy, Kevin R; Smith, Justin D; Vonesch, Sibylle C et al. (2018) Multiplexed precision genome editing with trackable genomic barcodes in yeast. Nat Biotechnol 36:512-520
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
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
Maki, Takahisa; Ogura, Naoto; Haber, James E et al. (2018) New insights into donor directionality of mating-type switching in Schizosaccharomyces pombe. PLoS Genet 14:e1007424
Botchkarev Jr, Vladimir V; Haber, James E (2018) Functions and regulation of the Polo-like kinase Cdc5 in the absence and presence of DNA damage. Curr Genet 64:87-96
Garbacz, Marta A; Lujan, Scott A; Burkholder, Adam B et al. (2018) Evidence that DNA polymerase ? contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae. Nat Commun 9:858
Eapen, Vinay V; Waterman, David P; Bernard, Amélie et al. (2017) A pathway of targeted autophagy is induced by DNA damage in budding yeast. Proc Natl Acad Sci U S A 114:E1158-E1167
Mehta, Anuja; Beach, Annette; Haber, James E (2017) Homology Requirements and Competition between Gene Conversion and Break-Induced Replication during Double-Strand Break Repair. Mol Cell 65:515-526.e3
Nakajima, Yuko; Haber, James E (2016) Chromosomes at loose ends. Nat Cell Biol 18:257-9

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