# The Effect of Subnuclear Compartmentalization on DNA Double-Strand Break Repair > **NIH NIH F31** · YALE UNIVERSITY · 2023 · $47,694 ## Abstract Project Summary/Abstract DNA double-stranded breaks (DSBs) represent a danger to genome stability. Cells can repair DSBs through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair pathway choice is critical because failure to choose the most faithful repair mechanism can lead to cancer. NHEJ is the direct ligation of the DSB ends, and as such it does not involve any repair templates. It is rapid, but error prone. In contrast, HR is the more faithful repair pathway that uses the sister chromatid as a template for repair and therefore is upregulated in S/G2 phases of the cell cycle. HR is initiated by the end resection of the DSB (creating 3’ single- stranded overhangs). It then proceeds with the loading of repair proteins, most importantly RAD51, onto the resulting ssDNA to allow the broken chromosome to strand invade a homologous template (ideally the sister chromatid). New synthesis and resolution complete homology-dependent repair. My project focuses on how HR is regulated post-resection, especially on how the recruitment of recombination factors is regulated in time and space. Previous work has shown that nuclear compartmentalization plays a role in regulating the loading of repair factors onto resected ssDNA through a mechanism involving RNA-DNA hybrids. Loading of repair factors is different in the two nuclear compartments: the euchromatic nuclear interior and the heterochromatic nuclear periphery. Specifically, in the nuclear interior, Rad52, which remodels RPA to Rad51 loading in yeast, is efficiently recruited to resected DSBs and is retained there during repair. By contrast, in DSBs tethered to the nuclear periphery, Rad52 loading is highly transient. My preliminary data suggest that Rad52 loading is antagonized at the nuclear periphery by hybridization of RNA with the resected ssDNA. Accordingly, reducing RNA/DNA hybrids by over-expression of RNase H partially restores Rad52 loading in DSBs at the nuclear periphery. This suggests a competitive mechanism between RNA-DNA hybrids and loading of pro-HR factors onto resected ssDNA at a DSB specifically at the nuclear periphery. My goal is to identify the source of the relevant RNAs, how they are processed, and how they affect DSB repair. My approach is to use a genetic model to investigate the loading of small RNAs onto resected ssDNA at asite-specific, inducible double- stranded break (DSB). I plan to use live-cell imaging, quantitative PCR, genetic perturbations, small RNA sequencing, and detection of RNA-DNA hybrids by DNA immunoprecipitation (DRIP using the S9.6 antibody). Further, I will use a previously designed imaging assay to observe the timing of Rad52 loading and resection progression in single cells and genetic repair outcome assays. For each of these approaches, I will use fission yeast strains that are genetically engineered to tether the DSB to the nuclear periphery and to overexpress RNase H2 (to degrade RNA-DNA hybrids). These results will shed light ... ## Key facts - **NIH application ID:** 10649464 - **Project number:** 5F31GM143917-02 - **Recipient organization:** YALE UNIVERSITY - **Principal Investigator:** Alyssa Laffitte - **Activity code:** F31 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2023 - **Award amount:** $47,694 - **Award type:** 5 - **Project period:** 2022-06-01 → 2024-05-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10649464 ## Citation > US National Institutes of Health, RePORTER application 10649464, The Effect of Subnuclear Compartmentalization on DNA Double-Strand Break Repair (5F31GM143917-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10649464. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*