# Biochemical and Mechanistic Insights into the Roles of Rad51 Paralogs in Homologous Recombination Repair

> **NIH NIH F31** · YALE UNIVERSITY · 2021 · $46,036

## Abstract

PROJECT SUMMARY/ABSTRACT
Understanding the molecular pathways disrupted in breast and ovarian cancer is vital to combating these
devastating diseases. Hereditary susceptibility to cancer can be caused by germline mutations in the BReast
CAncer susceptibility genes BRCA1 and BRCA2. The BRCA genes code for proteins involved in homologous
recombination (HR) repair of DNA Double-Stranded Breaks (DSBs) and the protection of reversed replication
forks. BRCA2, in particular, is vital to HR as it loads RAD51 onto single-stranded DNA (ssDNA) forming a
nucleoprotein filament. The five RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 are a
family of proteins with homology to RAD51, have been implicated in HR regulation, and germline mutations have
been linked to familial breast and ovarian cancer. The RAD51 paralogs form two different complexes in human
cells but their functions and stoichiometric relationships in HR remain uncharacterized decades after their
discovery. The BCDX2 complex (RAD51B/C/D/XRCC2) and the CX3 complex (RAD51C and XRCC3) have yet
to be successfully purified for biochemical assays. Genetic studies have made little progress as knockout cell
lines for any one of the paralogs are not viable. This proposal will utilize novel methods to overcome these
obstacles and better characterize the roles of the RAD51 paralogs in HR. The first approach will determine
whether specific domains mediate an interaction between RAD51 paralog proteins and BRCA2 or PALB2
(Partner and Localizer of BRCA2). Second, the RAD51 paralogs will be purified from human cells using a unique
and innovative protocol we have developed. The individual paralogs, as well as the two paralog complexes, will
be tested to determine if synergy exists with BRCA2 in stimulating RAD51-mediated DNA strand exchange. In a
complementary cell biological approach, I will use a conditional system to systematically deplete individual
RAD51 paralogs, and their respective complexes, to observe if RAD51 foci formation is compromised in
response to DNA damage. Finally, in collaboration with Dr. Eli Rothenberg at NYU, we will utilize his expertise
in super-resolution microscopy to test the hypothesis that the RAD51 paralog complexes are recruited to DSBs
to perform specific functions during HR. Using this technique, I will create a timeline of recruitment and retention
at DSBs in the context of other HR proteins. This spatiotemporal information will inform us at which step in HR
the paralogs are active and their spatial relationships at a single DSB. Upon completion of the research and
training fellowship, the RAD51 paralogs will be significantly more understood, and the applicant will have
received extensive training. The multidisciplinary mentoring team will prepare the applicant for research
independence and a successful career as a cancer researcher.

## Key facts

- **NIH application ID:** 10315679
- **Project number:** 1F31CA265150-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Jacob Garrett Thrasher
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $46,036
- **Award type:** 1
- **Project period:** 2021-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10315679

## Citation

> US National Institutes of Health, RePORTER application 10315679, Biochemical and Mechanistic Insights into the Roles of Rad51 Paralogs in Homologous Recombination Repair (1F31CA265150-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10315679. Licensed CC0.

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