Homologousrecombinationisachromosomerepairprocessthatplaysessentialrolesinmeiosis,the specializedcelldivisionthatproducesgametes.Defectsinmeioticrecombinationarealeadingcauseof infertility,pregnancylossandcongenitaldiseaseinhumans.Amajorgapinourunderstandingofmeiotic recombinationisthemechanismoftherecombination-associatedDNAsynthesis(RADS)thatisessentialto restorechromosomeintegrity.Thisknowledgegappersistsbecauseofinherentchallengestostudyingmeiotic RADSinvivo,inparticulartheessentialnatureofDNAreplicationfactors,theneedtostudyRADSinisolation fromchromosomalreplication,andtherequirementforspecialassaystomeasureRADS.Thesehurdleshave nowbeenovercomeusinganinnovativecombinationofchemical,real-timeandmoleculargeneticstoolsin buddingyeastthatenableacuteinactivationofessentialreplicationfactorsspecificallyduringrecombination, andmeasurementofdenovoDNAsynthesis.ThissystemutilizesanATP-analogsensitivealleleoftheCdc7 kinase(cdc7-as3)tosynchronizecellsafterS-phase,butbeforerecombinationisinitiated.Real-time inactivationofessentialreplicationfactorsisachievedusingtheauxin-inducibledegron(AID)system,which hasbeenrewiredandoptimizedforuseinmeioticcells.TomonitorRADS,newlysynthesizedDNAislabeled, isolatedandquantifiedusing5-ethynyl-2?-deoxyuridine(Edu)incorporation,biotin-azideclickchemistry, streptavidinpurificationandquantitativePCR(qPCR).Exploitingthesetools,thelong-termobjectivesofthis projectaretounderstandthenature,function,mechanismandregulationofRADS.Theseobjectiveswillbe pursuedthroughthreeaims.
Aim1 willdeterminetheroleofRADSforboththeDNAeventsofmeiotic recombinationandthechromosomaleventsofmeioticprophaseusingthecomprehensivebatteryof molecular,geneticandcytologicalassaysuniquelyavailableinbuddingyeast.
Aim2 willtestmodelsofRADS bydelineatingthereplicationfactorsinvolvedandsystematicallyanalyzingtheirroles.Complementarystudies inmousewillanalyzethelocalizationanddynamicsofreplicationfactorsatsitesofrecombination.
Aim3 will identifyandcharacterizefactorsinvolvedintherecruitmentofreplicationfactorstorecombinationsitesandthe regulationofRADS.Immunofluorescencecytologywillbeusedtomonitorchromosomaldynamicsof replicationfactorsanddeterminethegeneticrequirementsfortheirlocalization.ThetimingandextentofRADS willbeanalyzedinstrainsmutantforfactorspredictedtomodulateRADS,includingmeiosis-specific recombinationproteins,DNAhelicasesandtopoisomerases.Theresultsoftheseaimswillprovide unprecedentedinsightsintothemechanismandregulationofRADS,fillingamajorgapinourunderstandingof meioticrecombination.Thesefindingswillbegermanetounderstandingpathologiesassociatedwithhuman meiosis,andareexpectedtodefineparadigmsthatarebroadlyrelevantforchromosomerepair.

Public Health Relevance

TherepairofDNAbreaksbyhomologousrecombinationisessentialformeiosis,thecellularprocessthat producesspermandeggs.Defectsinmeioticrecombinationarealeadingcauseofinfertility,pregnancyloss andcongenitaldisease.Thisproposaladdressesacriticalgapinourunderstandingofmeioticrecombination, thenature,roleandmechanismoftheassociatedDNAsynthesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM074223-14
Application #
9604293
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Deatherage, James F
Project Start
2005-05-01
Project End
2022-07-31
Budget Start
2018-09-01
Budget End
2019-07-31
Support Year
14
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of California Davis
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Owens, Shannon; Tang, Shangming; Hunter, Neil (2018) Monitoring Recombination During Meiosis in Budding Yeast. Methods Enzymol 601:275-307
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 57:607-621
Hunter, Neil (2015) Meiotic Recombination: The Essence of Heredity. Cold Spring Harb Perspect Biol 7:
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
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
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
Lao, Jessica P; Hunter, Neil (2010) Trying to avoid your sister. PLoS Biol 8:e1000519
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

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