Double-stranded DNA breaks (DSBs) represent a critical genotoxic lesion that is induced directly or indirectly bymanyanti-tumoragentsincludingionizingradiation,interstrandcrosslinkingagents,topoisomerase-targeted drugs, and agents that lead to replication forks stalling. Homologous recombination (HR) is a central pathway of genome maintenance that repairs DSBs caused by these agents. Defects in HR have dual significance for cancer. They lead to genomic instability and predispose to cancer. On the other hand, HR defects cause specific cellular vulnerabilities that can be exploited therapeutically. The overarching goal is to elucidate the mechanisms of HR. In this award period, we focus on the central HR intermediate, the displacement loop (D- loop),whichiseithertargetedfordissociationbyanti-recombinationmechanismsorwhichisextendedbyDNA synthesistotransitionfurtherintheHRpathway.
The SpecificAims are: (1) Mechanism and significance of D-loop editing by Topoisomerase 3a?. Our published and unpublished workinS.cerevisiaeshowedthatTop3-Rmi1actasananti-recombinasebytargetingtheD-loopintermediate. Usingthetoolsandconceptsdevelopedinyeast,wewilldetermineinSubaim1Athebiochemicalmechanism involved using the yeast and human enzymes. In Subaim 1B, we will establish the significance of D-loop editingbyTOPOIIIa?inhumancells. (2) Determine the pathways of D-loop editing. Using a newly developed general assay based on the proximity ligation principle to physically detect D-loops, we will conduct pathway analysis to determine which enzymes and pathways act on D-loops. In Subaim 2A, we will complete the pathway analysis in the budding yeast. Most of the effort is dedicated to Subaim 2B to determine the human enzymes and pathways that regulateD-loopslevels. (3) Determine D-loop length and the factors controlling it. We have developed a novel assay to measure D-looplengthatthesingle-moleculelevel.
InSub aim3 A,wewillvalidatetheassayusingreconstitutedinvitro reactions with yeast and human recombination proteins. In Subaim 3B, we will define the mechanism, by which the motor protein Rdh54/Tid1 and its human homologs control D-loop length. In Subaim 3C, we will adapt this assay to human cells and determine D-loop length in human cells and test the human Rdh54/Tid1 paralogsfortheireffectonD-looplevelsandlength.

Public Health Relevance

Homologous recombination is a central pathway for maintaining genomic stability by repairing DNA double- strandedbreaksandothertypesofcomplexDNAdamagesuchasinterstrandcrosslinks.ThesetypesofDNA damages are induced by common DNA damage-inducing modalities in cancer treatment. The proposed work willcontributetoanimprovedmechanisticunderstandingofrecombinationalDNArepair,whichisfundamental toapplyingbiologicalapproachestoenhancetheefficacyandmitigatetheside-effectsofDNAdamage-based anti-cancertherapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058015-18
Application #
9764372
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Reddy, Michael K
Project Start
2000-01-01
Project End
2022-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
18
Fiscal Year
2019
Total Cost
Indirect Cost
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
Wright, William Douglass; Shah, Shanaya Shital; Heyer, Wolf-Dietrich (2018) Homologous recombination and the repair of DNA double-strand breaks. J Biol Chem 293:10524-10535
Piazza, Aurèle; Heyer, Wolf-Dietrich (2018) Multi-Invasion-Induced Rearrangements as a Pathway for Physiological and Pathological Recombination. Bioessays 40:e1700249
Piazza, Aurèle; Koszul, Romain; Heyer, Wolf-Dietrich (2018) A Proximity Ligation-Based Method for Quantitative Measurement of D-Loop Extension in S. cerevisiae. Methods Enzymol 601:27-44
Liu, Jie; Ede, Christopher; Wright, William D et al. (2017) Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase ?-extending D-loops. Elife 6:
Muñoz-Galván, Sandra; Tous, Cristina; Blanco, Miguel G et al. (2017) Correction for Muñoz-Galván et al., ""Distinct Roles of Mus81, Yen1, Slx1-Slx4, and Rad1 Nucleases in the Repair of Replication-Born Double-Strand Breaks by Sister Chromatid Exchange"". Mol Cell Biol 37:
Piazza, Aurèle; Wright, William Douglass; Heyer, Wolf-Dietrich (2017) Multi-invasions Are Recombination Byproducts that Induce Chromosomal Rearrangements. Cell 170:760-773.e15
Crawley, Jacqueline N; Heyer, Wolf-Dietrich; LaSalle, Janine M (2016) Autism and Cancer Share Risk Genes, Pathways, and Drug Targets. Trends Genet 32:139-146
Spies, Julian; Waizenegger, Anja; Barton, Olivia et al. (2016) Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability. Mol Cell 62:903-917
McVey, Mitch; Khodaverdian, Varandt Y; Meyer, Damon et al. (2016) Eukaryotic DNA Polymerases in Homologous Recombination. Annu Rev Genet 50:393-421
Ganai, Rais A; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich et al. (2016) Strand displacement synthesis by yeast DNA polymerase ?. Nucleic Acids Res 44:8229-40

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