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Determining the Role of Replication Protein A in Polymerase Theta-Mediated End-Joining

Award Number: F30CA298183
ORGANIZATION: NATIONAL CANCER INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 04/01/2025
PERIOD OF PERFORMANCE END DATE: 03/31/2029

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Determining the Role of Replication Protein A in Polymerase Theta-Mediated End-Joining - PROJECT SUMMARY Many frontline cancer treatments, including radiation therapy and several chemotherapeutics, work by inducing double-strand breaks (DSBs) since cancer cells are typically more vulnerable to DSBs than healthy cells. Thus, understanding the complex pathways cancer cells use to repair DSBs is critical to understanding and addressing resistance to these therapeutic agents, which almost invariably arises. Deficiencies in the high fidelity DSB repair pathway homologous recombination promote tumorigenesis. The resulting cancers are dependent on a mutagenic DSB repair pathway called polymerase theta-mediated end-joining (TMEJ). Polymerase theta (Polθ) is a multifunctional enzyme consisting of an N-terminal helicase domain, disordered linker domain and C-terminal polymerase domain. Polθ is essential for TMEJ, but the other factors involved in this pathway remain poorly defined. While genetic screens have revealed several non-essential proteins that may play a role in TMEJ, these screens are incapable of identifying essential factors that interact with Polθ to promote TMEJ. In this proposal, I will determine the role of the essential, eukaryotic single strand DNA-binding protein, replication protein A (RPA), in TMEJ. Based on partial knockdown in cells, RPA was initially thought to inhibit TMEJ.1 However, when I depleted RPA from a cell-free TMEJ assay in Xenopus egg extracts, end- joining was abolished. TMEJ was rescued with recombinant RPA, demonstrating that RPA is essential for TMEJ. The deep learning tool Alpha-Fold Multimer predicts that RPA and Polθ interact via two interfaces — one on RPA1 and one on RPA2. When I mutated these interfaces on RPA, replication was unaffected but TMEJ was inhibited. Mutating the corresponding residues on Polθ also abolished TMEJ. I hypothesize that these interfaces are necessary for Polθ recruitment and annealing of microhomologous regions. In Aim 1, I will test the hypothesis that RPA1 binds and recruits Polθ and that this allows Polθ to bridge two non-homologous strands. In Aim 2, I will test the hypothesis that RPA2 promotes TMEJ by stimulating the helicase domain of Polθ to simultaneously bind DNA, anneal two distinct strands and destabilize and remove RPA. Understanding the role of RPA in TMEJ will provide fundamental insight into how Polθ interacts with other proteins to repair DSBs both in health and disease.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $42,796 )
2026	2026	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Cancer Research Manpower	001	2	4/24/2026	NON-COMPETING CONTINUATION	$576
2026	2026	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Cancer Research Manpower	000	2	2/24/2026	NON-COMPETING CONTINUATION	$42,220
														Subtotal = $42,796

Issue Date FY: 2025 ( Subtotal = $42,220 )
2025	2025	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Cancer Research Manpower	001	1	2/14/2025	NEW	$0
2025	2025	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Cancer Research Manpower	002	1	7/8/2025	NEW	$564
2025	2025	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Cancer Research Manpower	000	1	2/14/2025	NEW	$41,656
														Subtotal = $42,220

Grand Total All Awards = $85,016

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Determining the Role of Replication Protein A in Polymerase Theta-Mediated End-Joining

Award Number: F30CA298183

ORGANIZATION: NATIONAL CANCER INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 04/01/2025

PERIOD OF PERFORMANCE END DATE: 03/31/2029

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