# Regulation of DNA double-strand break repair pathway choice

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2023 · $466,640

## Abstract

Project Summary
The general goal of the proposed research is to define the mechanistic underpinning for the synthetic lethality
network between a deficiency in homologous recombination (HR) and defects in single-strand annealing
(RAD52) or POLq-mediated end-joining (TMEJ). Exciting preliminary data revealed that BRCA2 and RAD52
delay the repair of S-phase-associated DNA double-stranded breaks (DSB) by TMEJ until M-phase. This
regulation avoids TMEJ-mediated chromosomal rearrangements of one-sided DSBs that are produced by
replication fork breakage. Our approach combines innovative cell cycle phase resolved imaging of DNA repair
proteins and DNA damage markers with mechanistic biochemical analysis using purified human proteins in
reconstituted reactions. Our results will have potential translational implications for the clinical application of
newly developed RAD52 and POLq inhibitors for the treatment of HR-deficient tumors with respect to application
protocols, patient selection, and use of DNA damage response checkpoint inhibitors as well as the response to
poly(ADP-ribose) polymerase inhibition.
The Specific Aims are:
1. Define the mechanism of action of BRCA2 in DSB repair pathway control. We will test the model that the
 DNA binding properties of BRCA2 are critical for TMEJ inhibition. In Aim 1A, we conduct foundational studies
 to determine the fundamental DNA binding properties of full-length BRCA2. In Aim 1B, we will define which
 domains of BRCA2 are required for TMEJ inhibition in cells. This combination of cell-based and biochemical
 studies will define the functions and regions of BRCA2 that are required for TMEJ inhibition.
2. Define the mechanism of TMEJ inhibition by BRCA2 and RAD52. BRCA2 and RAD52 employ two different
 modes to inhibit the DNA polymerase activity of POLq which may affect additional reaction steps in the TMEJ
 process. We will reconstitute TMEJ in vitro with purified proteins to determine the mechanisms by which
 BRCA2 (Aim 2A) and RAD52 (Aim 2B) inhibit TMEJ. We will test inhibition of the overall TMEJ reaction and
 individual steps including 1) DNA binding, 2) end-alignment, and 3) DNA synthesis. Analysis of wild type and
 catalytic mutants of POLq will be conducted in vitro and in cells.
3. Define which HR defects are susceptible to RAD52 loss of function. It is an open question whether loss
 of RAD52 will lead to POLq-mediated chromosome fusions and lethality in all HR-deficient backgrounds (Aim
 3A) or all BRCA2 mutants (Aim 3B). Our preliminary studies suggest a model that loading of BRCA2 is the
 critical step to limit TMEJ to M-phase and that HR defects past this step are not affected by RAD52 inhibition.

## Key facts

- **NIH application ID:** 10656805
- **Project number:** 1R01CA273911-01A1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Wolf-Dietrich Heyer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $466,640
- **Award type:** 1
- **Project period:** 2023-07-01 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10656805

## Citation

> US National Institutes of Health, RePORTER application 10656805, Regulation of DNA double-strand break repair pathway choice (1R01CA273911-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10656805. Licensed CC0.

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