Homologous recombination occurs when a broken or damaged chromosome uses a homologous chromosome as repair template. Recombination underpins DNA replication and genome stability, and is essential for chromosome segregation during meiosis. Our long-term goal is to understand the mechanism and regulation of recombination. The fundamental recombination reaction is the formation of Joint Molecule (JM) intermediates via pairing and strand-exchange between a broken chromosome and a homologous template. A multiplicity of DNA nucleases and helicases function during every step of recombination and assigning in vivo functions to specific enzymes, and understanding how they interact during recombination remain challenging issues. This proposal will investigate the in vivo roles of nucleases and helicases in JM metabolism.
Specific Aim 1. To Characterize the Pathways of Holliday Junction Resolution. We have identified five activities responsible for essentially all JM resolution and crossing-over during meiosis. These include mismatch repair factors, Exo1, Mlh1 and Mlh3;XPF-family nuclease, Mus81-Mms4;Slx4, which forms two distinct nuclease complexes;the recently identified HJ resolvase, Yen1;and RecQ helicase, Sgs1, which functions in a complex to dissociate dHJs rather than resolve them. Molecular and genetic approaches will be used to assign specific roles to these activities in vivo and test specific models of meiotic JM resolution. An assays system has been developed that, for the first time, allows detection of JMs formed in vivo during mitotic DSB-repair. This unique tool will be used to determine the roles of JM resolving factors in mitotic DSB-repair.
Specific Aim 2. To Determine the Roles of DNA Helicases in Regulating Joint Molecule Metabolism. Three DNA helicases, Sgs1, Srs2 and Mph1 appear to function independently to suppress crossing-over during mitotic DSB-repair. However, during meiosis, these anti-crossover factors must be inhibited at sites of crossing-over, but may be required to complete non-crossover recombination. Molecular assays will be used to determine the roles of these helicases in regulating JM formation during meiosis and during mitotic DSB-repair. A specific model of meiotic JM formation and the roles of helicases in this process will be examined. We will also test the idea that Srs2 is prevented from disrupting filaments of RecA proteins, Rad51 and Dmc1, during meiosis by the so-called "mediator" proteins. Finally, we will examine the molecular roles of two additional helicases: Hrq1, a recently identified fungal homolog of RecQ4, which is mutated in Rothmund-Thomson syndrome;and the helicase/nuclease Dna2, which is defective for crossing-over during mitotic DSB-repair Defective recombination is associated with infertility, pregnancy miscarriage and genetic disease. In somatic cells is especially relevant for cancer. An understanding of the molecular processes of homologous recombination will help us better understand the etiology of these disorders.

Public Health Relevance

Homologous recombination is required for sexual reproduction and chromosome repair. Defects in this process are linked to human infertility, miscarriage and genetic diseases, especially cancer. A greater understanding of the mechanism and regulation of homologous recombination will help us better understand the etiology of these diseases and design novel therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074223-09
Application #
8463555
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Janes, Daniel E
Project Start
2005-05-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
9
Fiscal Year
2013
Total Cost
$276,168
Indirect Cost
$89,875
Name
University of California Davis
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Hunter, Neil (2015) Meiotic Recombination: The Essence of Heredity. Cold Spring Harb Perspect Biol 7:
Tang, Shangming; Wu, Michelle Ka Yan; Zhang, Ruoxi et al. (2015) Pervasive and essential roles of the Top3-Rmi1 decatenase orchestrate recombination and facilitate chromosome segregation in meiosis. Mol Cell 57:607-21
Lao, Jessica P; Cloud, Veronica; Huang, Chu-Chun et al. (2013) Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation. PLoS Genet 9:e1003978
Copsey, Alice; Tang, Shangming; Jordan, Philip W et al. (2013) Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions. PLoS Genet 9:e1004071
Sasanuma, Hiroyuki; Tawaramoto, Maki S; Lao, Jessica P et al. (2013) A new protein complex promoting the assembly of Rad51 filaments. Nat Commun 4:1676
Lao, Jessica P; Tang, Shangming; Hunter, Neil (2013) Native/Denaturing two-dimensional DNA electrophoresis and its application to the analysis of recombination intermediates. Methods Mol Biol 1054:105-20
Zakharyevich, Kseniya; Tang, Shangming; Ma, Yunmei et al. (2012) Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase. Cell 149:334-47
Zakharyevich, Kseniya; Ma, Yunmei; Tang, Shangming et al. (2010) Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions. Mol Cell 40:1001-15
Lao, Jessica P; Hunter, Neil (2010) Trying to avoid your sister. PLoS Biol 8:e1000519
Bzymek, Malgorzata; Thayer, Nathaniel H; Oh, Steve D et al. (2010) Double Holliday junctions are intermediates of DNA break repair. Nature 464:937-41

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