The long-term objective of this project is to understand the molecular mechanism of homologous recombination initiation during meiosis. Recombination is essential for meiosis in most sexually reproducing organisms, including humans, because it generates genetic diversity and ensures the accurate segregation of homologous chromosomes. In S. cerevisiae, meiotic recombination initiates via the formation of DNA double-strand breaks (DSBs) through a transesterification reaction catalyzed by Spo11, a member of a widely conserved family that includes meiotic recombination proteins from higher eukaryotes and type II topoisomerases of archaebacteria. This study will use Spo11 as the focal point to investigate the molecular mechanism of DSB formation.
The Specific Aims are: l. To identify proteins that interact with Spo11. At least nine gene products in addition to Spo11 are required for DSB formation, suggesting that physical interactions between Spo11 and other proteins are important for Spo11 function. These interactions will be identified using several complementary approaches: a) Spo11-containing complexes will be affinity-purified from extracts of meiotic cells, and the protein components will be identified by mass spectrometric tryptic peptide fingerprinting. b) Proteins required for DSB formation will be coexpressed with Spo11 in vitro and assayed for complex formation. And c) yeast two-hybrid screens with Spoil as bait will be carried out. 2. To characterize the activities of Spoil and the proteins that interact with Spo11. Spo11 will be over-expressed and purified, and will be examined for DNA binding, DNA cleavage, and topoisomerase activities. Proteins found to interact with Spo11 will also be overexpressed and purified. Their activities and their effects on the activities of Spo11 will be characterized. 3. To develop an in vitro assay system for DSB formation. Recent evidence indicates that chromatin and higher order structures of meiotic chromosomes influence the locations and frequencies of DSBs. To begin to address the molecular basis of these effects, a biochemically accessible system that uses whole meiotic chromosomes as substrates for DSB formation will be developed. Conditions have been identified in which nuclei isolated from wild-type meiotic cells are competent to form new DSBs in vitro. This system will be optimized and characterized, then the biochemical components will be dissected by fractionation of wild- type nuclei, and by adaptation for in vitro complemenation of nuclei isolated from DSB-defective mutant cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058673-02
Application #
6138688
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Anderson, Richard A
Project Start
1999-01-01
Project End
2003-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
2
Fiscal Year
2000
Total Cost
$303,586
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Mimitou, Eleni P; Keeney, Scott (2018) S1-seq Assay for Mapping Processed DNA Ends. Methods Enzymol 601:309-330
Marsolier-Kergoat, Marie-Claude; Khan, Md Muntaz; Schott, Jonathan et al. (2018) Mechanistic View and Genetic Control of DNA Recombination during Meiosis. Mol Cell 70:9-20.e6
Kniewel, Ryan; Murakami, Hajime; Liu, Yan et al. (2017) Histone H3 Threonine 11 Phosphorylation Is Catalyzed Directly by the Meiosis-Specific Kinase Mek1 and Provides a Molecular Readout of Mek1 Activity in Vivo. Genetics 207:1313-1333
Mimitou, Eleni P; Yamada, Shintaro; Keeney, Scott (2017) A global view of meiotic double-strand break end resection. Science 355:40-45
Lam, Isabel; Mohibullah, Neeman; Keeney, Scott (2017) Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast. Methods Mol Biol 1471:51-98
Mohibullah, Neeman; Keeney, Scott (2017) Numerical and spatial patterning of yeast meiotic DNA breaks by Tel1. Genome Res 27:278-288
Barchi, Marco; Cohen, Paula; Keeney, Scott (2016) Special issue on ""recent advances in meiotic chromosome structure, recombination and segregation"". Chromosoma 125:173-5
Lange, Julian; Yamada, Shintaro; Tischfield, Sam E et al. (2016) The Landscape of Mouse Meiotic Double-Strand Break Formation, Processing, and Repair. Cell 167:695-708.e16
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
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

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