The objectives of this research are to understand the mechanism of meiotic recombination and to determine how this process is coordinated with other events of meiotic prophase. The studies focus on yeast Spo11 (the protein that makes the double-strand breaks (DSBs) that initiate meiotic recombination) and the proteins that interact with it. In the new grant period, experiments are proposed to continue studying the mechanisms of DSB formation and repair.
The specific aims are: 1. To determine mechanisms that regulate DSB formation via modification of Mer2. Two cell cycle regulatory kinases, cyclin-dependent kinase and Cdc7, converge on the Mer2 protein to control DSB formation. Molecular details of Mer2 regulation by these kinases will be defined by determining patterns of Mer2 localization on chromosomes and determining when and where Mer2 phosphorylation occurs. These studies will also test the hypothesis that regulation by Cdc7 coordinates timing of DSB formation with DNA replication. 2. To biochemically characterize DSB proteins and multiprotein complexes. Spo11 requires activities of nine proteins in order to generate meiotic DSBs. These DSB proteins can be divided into several distinct functional subgroups based on pairwise interactions among them, chromosomal localization patterns, dependencies for chromatin association, and other behaviors. The biochemical nature of these subgroups and the interactions among them remain unclear, however. To provide more detailed understanding of these issues, DSB proteins will be affinity-purified from meiotic extracts, associated factors will be identified, and the architecture and biochemical activities of stable multiprotein complexes will be characterized. Structure- function analysis is also proposed for Rec102-Rec104, one of the DSB protein subgroups. 3. To define the genetic and region-specific control of crossover homeostasis. Cells have the ability to maintain high levels of crossovers when DSB frequencies are reduced. This phenomenon is referred to as """"""""crossover homeostasis"""""""" and has been proposed to be mechanistically related to interference and other processes that control the number and distribution of crossovers. This hypothesis will be tested by determining whether mutants with known defects in crossover interference show parallel defects in crossover homeostasis. Studies are also proposed to test the hypothesis that crossover homeostasis and the crossover-noncrossover decision vary from location to location on chromosomes, and to develop new assays for crossover and noncrossover recombination that do not require the generation of viable meiotic products. Abnormal chromosome numbers in eggs or sperm cause developmental disabilities or spontaneous abortion. These abnormalities often arise because of improper separation of chromosomes caused by defects in meiotic homologous recombination. This project will address fundamental questions about the mechanism and control of recombination.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058673-13
Application #
8025936
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Janes, Daniel E
Project Start
1999-01-01
Project End
2012-03-31
Budget Start
2011-01-01
Budget End
2012-03-31
Support Year
13
Fiscal Year
2011
Total Cost
$377,357
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Barchi, Marco; Cohen, Paula; Keeney, Scott (2016) Special issue on ""recent advances in meiotic chromosome structure, recombination and segregation"". Chromosoma 125:173-5
Lam, Isabel; Keeney, Scott (2015) Nonparadoxical evolutionary stability of the recombination initiation landscape in yeast. Science 350:932-7
Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel et al. (2015) The kinetochore prevents centromere-proximal crossover recombination during meiosis. Elife 4:
Zhu, Xuan; Keeney, Scott (2015) High-Resolution Global Analysis of the Influences of Bas1 and Ino4 Transcription Factors on Meiotic DNA Break Distributions in Saccharomyces cerevisiae. Genetics 201:525-42
Chen, Xiangyu; Suhandynata, Ray T; Sandhu, Rima et al. (2015) Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis. PLoS Biol 13:e1002329
Lam, Isabel; Keeney, Scott (2015) Mechanism and regulation of meiotic recombination initiation. Cold Spring Harb Perspect Biol 7:a016634
Murakami, Hajime; Keeney, Scott (2014) DDK links replication and recombination in meiosis. Cell Cycle 13:3621-2
Thacker, Drew; Mohibullah, Neeman; Zhu, Xuan et al. (2014) Homologue engagement controls meiotic DNA break number and distribution. Nature 510:241-6
Keeney, Scott; Lange, Julian; Mohibullah, Neeman (2014) Self-organization of meiotic recombination initiation: general principles and molecular pathways. Annu Rev Genet 48:187-214
Murakami, Hajime; Keeney, Scott (2014) Temporospatial coordination of meiotic DNA replication and recombination via DDK recruitment to replisomes. Cell 158:861-73

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