This proposal continues our study of the mechanisms of homologous recombination in the model system, the budding yeast Saccharomyces cerevisiae. The mating-type (MAT) locus is induced to switch to the opposite mating-type through the repair of a double-strand break (DSB). Inducible expression of the HO endonuclease gene can be used to analyze MAT switching and other DSB repair events by physical monitoring of DNA undergoing recombination. The relationship between gene conversion and break-induced replication will be studied, including analysis of protein and cell cycle requirements. Density-transfer experiments will be used to determine the extent and location of newly synthesized DNA during recombination. Chromatin immunoprecipitation will be used to analyze the roles of specific recombination proteins. Genetic and molecular biological approaches will also be used to analyze the histone modification of chromatin during recombination and the reassembly of chromatin when recombination is complete. Mutations affecting control of crossing-over will be analyzed to understand how most gene conversions in mitotic cells occur without exchange, avoiding loss of heterozygosity and chromosome rearrangements. Break-induced replication will be further characterized to understand its role in maintaining telomeres in the absence of telomerase and in generating nonreciprocal translocations. The MAT switching system will also be used to learn about the regulation of accessibility of chromosome regions for recombination events. The choice of one of two distant donor sequences, HML or HMR, for recombination with MAT is controlled by a locus control region, the recombination enhancer (RE). Emphasis is placed on determining how the entire left arm of chromosome III is sequestered in MATa cells and how RE and its associated forkhead Fhkl protein makes the arm """"""""hot"""""""" for recombination. Each of these subjects has high relevance for our understanding of the ways broken chromosomes are repaired in humans and how mutations in a number of genes that facilitate recombination lead to an increased predisposition to cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM020056-33
Application #
6892322
Study Section
Genetics Study Section (GEN)
Program Officer
Portnoy, Matthew
Project Start
1976-01-01
Project End
2007-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
33
Fiscal Year
2005
Total Cost
$601,736
Indirect Cost
Name
Brandeis University
Department
Type
Organized Research Units
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
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Tsabar, Michael; Waterman, David P; Aguilar, Fiona et al. (2016) Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair. Genes Dev 30:1211-24

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