The objective of this research is to understand the mechanism of mammalian meiotic recombination. Crossovers occur nonrandomly in the genome, forming preferentially within 1-2 kb "hotspots" where SPO11 protein forms double-strand breaks (DSBs). Understanding hotspot activity and distribution is important for understanding the role of recombination in chromosome segregation. Mouse meiosis is an ideal system for these studies because of its evolutionary similarity to human meiosis. Factors that contribute to hotspot activity in mouse will be characterized, and a new method for identifying recombination hotspots will be developed.
The specific aims are: 1. To identify factors that contribute to variation in crossover hotspot activity. Little is known about the molecular determinants of hotspot function in mammals. To address this issue, we will examine effects of sex, strain background, and local sequence differences on recombination frequencies at selected hotspots. 2. To test for sex-specific and chromosome region-specific variation in the crossover vs. noncrossover decision. Crossovers are only ~10% of the recombination products in a meiotic cell-the majority of DSBs are repaired to give noncrossover products. We have proposed that non random crossover placement arises in part from programmed deviation from the genome- average crossover:noncrossover ratio and that this deviation accounts for differences in crossover position between males and females. We will test this hypothesis by comparing relative frequencies of crossovers and noncrossovers between male and female at hotspots within chromosomal regions with sexually dimorphic crossover rates. We will similarly test whether hotspots at different genomic positions in males vary with respect to the crossover:noncrossover ratio. 3. To develop a new method to directly identify DSB hotspots using SPO11-associated oligonucleotide sequences. SPO11 protein cleaves DNA via a topoisomerase-like reaction that leaves SPO11 covalently attached to the 52 termini of the DSB. We recently demonstrated that these protein-associated DSBs are processed by an endonucleolytic mechanism that releases SPO11 covalently bound to a short oligonucleotide. We will exploit this finding to identify DSB hotspots by sequencing the SPO11-associated DNA. We will use yeast meiosis as a model system for proof of principle and to extend this methodology to high-throughput methods to map and quantify large numbers of hotspots across the genome. We will then extend these studies to identify DSB hotspots in the mouse.

Public Health Relevance

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
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD053855-05
Application #
8459531
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Taymans, Susan
Project Start
2009-05-09
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2013
Total Cost
$354,836
Indirect Cost
$167,686
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Cole, Francesca; Baudat, Frédéric; Grey, Corinne et al. (2014) Mouse tetrad analysis provides insights into recombination mechanisms and hotspot evolutionary dynamics. Nat Genet 46:1072-80
Keeney, Scott; Lange, Julian; Mohibullah, Neeman (2014) Self-organization of meiotic recombination initiation: general principles and molecular pathways. Annu Rev Genet 48:187-214
de Boer, Esther; Jasin, Maria; Keeney, Scott (2013) Analysis of recombinants in female mouse meiosis. Methods Mol Biol 957:19-45
Dowdle, James A; Mehta, Monika; Kass, Elizabeth M et al. (2013) Mouse BAZ1A (ACF1) is dispensable for double-strand break repair but is essential for averting improper gene expression during spermatogenesis. PLoS Genet 9:e1003945
Pan, Jing; Sasaki, Mariko; Kniewel, Ryan et al. (2011) A hierarchical combination of factors shapes the genome-wide topography of yeast meiotic recombination initiation. Cell 144:719-31
Daniel, Katrin; Lange, Julian; Hached, Khaled et al. (2011) Meiotic homologue alignment and its quality surveillance are controlled by mouse HORMAD1. Nat Cell Biol 13:599-610
Lange, Julian; Pan, Jing; Cole, Francesca et al. (2011) ATM controls meiotic double-strand-break formation. Nature 479:237-40
Cole, Francesca; Keeney, Scott; Jasin, Maria (2010) Comprehensive, fine-scale dissection of homologous recombination outcomes at a hot spot in mouse meiosis. Mol Cell 39:700-10
Sasaki, Mariko; Lange, Julian; Keeney, Scott (2010) Genome destabilization by homologous recombination in the germ line. Nat Rev Mol Cell Biol 11:182-95
Cole, Francesca; Keeney, Scott; Jasin, Maria (2010) Evolutionary conservation of meiotic DSB proteins: more than just Spo11. Genes Dev 24:1201-7

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