Abstract 526: The role of ssDNA in alternative DNA DSB repair and the opportunity for therapeutic intervention.

Abstract DNA double-stranded breaks (DSBs) create genomic instability, a driving factor in cancer development. Targeting DNA repair is a pivotal strategy in cancer therapy by exploiting synthetic lethal interactions with PARP inhibitors in patients deficient in homologous recombination (HR) DSB repair. Despite these advances, the clinical outcomes and recurrence rate for these patients have remained relatively stagnant. Recurrence is a direct result of treatment resistance mechanisms which can include reactivation of HR or the use of alternative end-joining (alt-EJ) DSB repair pathways. Theta-Mediated End Joining (TMEJ) and Single-Strand Annealing (SSA) can be employed to maintain genome stability and elevated expression of proteins involved in these pathways, including PolQ and XPF, have been reported in ovarian and lung cancer. SSA and TMEJ repair pathways involve common steps of resection, homology searching/annealing, and DNA synthesis. However, protection and processing of the ssDNA intermediates has not been addressed. Replication Protein A (RPA) is the major single-stranded DNA (ssDNA) binding protein involved in replication and repair and has been implicated in both TMEJ and SSA repair pathways though definitive involvement and the putative mechanisms have not been elucidated. We propose that RPA impacts TMEJ and SSA dependent DNA DSB repair via binding to ssDNA intermediates. The impact of RPA activity on TMEJ and SSA was assessed via genetic knockdown (KD) of RPA using siRNA in HEK293T and H1299 cell lines. Following siRNA KD, TMEJ and SSA repair activity were measured using a repair pathway specific dual luciferase extrachromosomal reporter assay. Results demonstrate that RPA stimulates both TMEJ and SSA activity as loss of RPA activity resulted in a ≥50% reduction repair activity for both pathways. Furthermore, a qPCR based extrachromosomal reporter assay requiring an additional 25bp of synthesis for complete repair was also reliant on RPA. Interestingly, treatment of RPA KD cells with ART558, a PolQ inhibitor, had less of an impact compared the control cells suggesting that cells can use a PolQ-independent mechanism of TMEJ for repair under certain conditions. Additionally, the impact of XPF-ERCC1 activity on SSA repair was considered as XPF-ERCC1’s major function is to cleave 3’ ssDNA overhangs, a key intermediate that must be processed in SSA. The impact was assessed via SSA repair activity in H1299 XPF-ERCC1 genetic knockout cells compared to the H1299 Cas9 control cells. Loss of XPF-ERCC1 activity resulted in approximately a 6-fold decrease in SSA activity indicating that SSA repair heavily relies of XPF-ERCC1 for ssDNA processing. Collectively, these data establish that RPA and XPF have a stimulatory role in TMEJ and SSA repair. The insights gained from this research can be used to better understand how certain types of cancer modify their DNA repair mechanisms to enhance their chances of survival. Citation Format: Jessica Lynn Kersey, John J. Turchi. The role of ssDNA in alternative DNA DSB repair and the opportunity for therapeutic intervention abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 526.