Regulation of DNA double-strand break repair pathway choice

Active Project Grant Awarded Last action

Award ID
R01CA273911
Recipient
Awarding agency
Obligated
$2,824,252

Award facts

Assistance listing
93.393 Cancer Cause and Prevention Research
Completion date 66% complete
Place of performance
Davis, CA, USA

Award details come from USAspending.gov.

Description

As the agency reported it.

Abstract
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.

Transactions

Action Obligated Date
Amendment $453,903
Amendment $453,903
Amendment $120,750
Amendment $431,208
Amendment $431,208
Amendment $466,640
Original award $466,640

Subawards

Subrecipient Amount Awarded
Sloan-Kettering Institute For Cancer Research Regulation of DNA double-strand break repair pathway choice $31,032
Technische Universität Darmstadt Scope of work – Cell Biology As part of Aim 1B, we will conduct cell biological experiments to identify which functions of BRCA2 are required for… $85,158

Quick answers

Who received award R01CA273911?

R01CA273911 was awarded to University Of California, Davis by National Cancer Institute (National Institutes of Health) on July 1, 2023.

How much is award R01CA273911 worth?

$2,824,252 has been obligated on R01CA273911.

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