Homologous recombination during meiosis is essential for genome integrity in the germ line, but is also a powerful determinant of genome diversity, evolution, and (when mistakes occur) instability. Meiotic recombination is initiated by double-strand breaks (DSBs) made by the Spo11 protein. This proposal addresses molecular mechanisms underlying DSB formation and recombination in the budding yeast, S. cerevisiae.
Aims are: 1) To define regulatory mechanisms that ensure that DSBs form after DNA replication. A working model will be tested, in which physical association of a cell cycle regulatory kinase with the replisome targets Mer2 protein for phosphorylation specifically in chromatin that has been replicated. The genome-wide relationship between replication and DSB formation will also be explored. 2) To understand factors that determine DSB distributions. Different genomic regions show different propensity for DSB formation, with this "DSB landscape" shaped by combinatorial and hierarchical action of many factors. Detailed understanding of these factors is lacking. A novel method for genome-wide mapping of DSBs at nucleotide resolution will be used to determine the contribution of local chromatin structure and of proteins involved in higher-order chromosome folding (cohesins and chromosome axis proteins). Additionally, DSB maps in divergent wild-type laboratory strains will test how DSB distributions vary with genetic background. 3) To determine the relationship between DSB location and gene conversion tracts. Classical tetrad analysis will be combined with state-of-the-art genotyping methods and novel physical assays to map gene conversion tracts with high spatial and quantitative precision around a set of selected DSB hotspots. Comparing these maps to high resolution DSB maps will test predictions of different recombination models. 4) To determine how DSBs in repetitive sequences contribute to genome instability. Recombination between dispersed homologous DNA segments can lead to chromosome rearrangements. Such non-allelic recombination has been extensively studied in yeast meiosis, but usually with artificial repeats. Here, the occurrence of DSBs within and near natural repeats (retrotransposon Ty elements) will be determined using novel genome-wide mapping methods and physical assays. In addition, a new method for detecting and quantifying crossing over between non- allelic Ty elements will be used to determine the extent to which DSBs within Tys contribute to gross chromosomal rearrangements.

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
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058673-16
Application #
8622204
Study Section
Special Emphasis Panel (ZRG1-GGG-R (03))
Program Officer
Janes, Daniel E
Project Start
1999-01-01
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
16
Fiscal Year
2014
Total Cost
$405,990
Indirect Cost
$184,016
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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
Murakami, Hajime; Keeney, Scott (2014) Temporospatial coordination of meiotic DNA replication and recombination via DDK recruitment to replisomes. Cell 158:861-73
Thacker, Drew; Mohibullah, Neeman; Zhu, Xuan et al. (2014) Homologue engagement controls meiotic DNA break number and distribution. Nature 510:241-6
Fowler, Kyle R; Sasaki, Mariko; Milman, Neta et al. (2014) Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome. Genome Res 24:1650-64
Keeney, Scott; Lange, Julian; Mohibullah, Neeman (2014) Self-organization of meiotic recombination initiation: general principles and molecular pathways. Annu Rev Genet 48:187-214
Sasaki, Mariko; Tischfield, Sam E; van Overbeek, Megan et al. (2013) Meiotic recombination initiation in and around retrotransposable elements in Saccharomyces cerevisiae. PLoS Genet 9:e1003732
Tischfield, Sam E; Keeney, Scott (2012) Scale matters: the spatial correlation of yeast meiotic DNA breaks with histone H3 trimethylation is driven largely by independent colocalization at promoters. Cell Cycle 11:1496-503
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
Thacker, Drew; Lam, Isabel; Knop, Michael et al. (2011) Exploiting spore-autonomous fluorescent protein expression to quantify meiotic chromosome behaviors in Saccharomyces cerevisiae. Genetics 189:423-39

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