RNA's Role in Double-Strand Break Repair

/ABSTRACT Double-strand breaks (DSBs) are a highly toxic form of DNA damage. The accurate and preferred DSB repair pathway is homologous recombination (HR), which uses the sister chromatid as a repair template during S/G2 cell cycle stages. Alternatively, the error-prone nonhomologous end joining (NHEJ) ligates broken ends together primarily during G1. Recently, data in yeast suggest that RNA can be used as a template for DNA DSB repair through reverse transcriptase (RT) activity. While human cells have shown that RNA transcription promotes the recruitment of DNA repair proteins to DSBs, there is no evidence that RNA is directly templating repair. This has so far been difficult to study due to a lack of assays to measure this scarless repair pathway. Therefore, we developed a fluorescence-based BFP-to-GFP conversion reporter that measures repair of a DSB in the BFP gene via a single-stranded donor oligonucleotide coding for GFP. From this reporter, ribonucleotides within a DNA donor could template DSB repair in human cells, necessitating the use of a reverse transcriptase for this RNA-templated DSB repair (RT-DSBR). Through a CRISPR-Cas9 screen, we identified the involvement of Polymerase Zeta (PolZ). We validated the role of PolZ in RT-DSBR through the BFP-to-GFP assay as well as a sequencing-based assay using a pure RNA donor with an insertion signature. Therefore, the objective of my project is to unveil how PolZ and RNA work together to drive RT-DSBR in human cells. I hypothesize that mRNA can template DSB repair at highly transcribed regions and that PolZ is the reverse transcriptase that mediates this activity. The first part of my research will investigate the mechanistic basis of PolZ as an RT during RT- DSBR (Aim 1). I will identify the subunits of PolZ necessary for RT-DSBR activity via a pure RNA donor assay and use structure-function analysis to confirm its reverse transcriptase catalytic domain (Aim 1.1). I will next use immunofluorescence to visualize if PolZ recruitment to breaks is dependent on its scaffolding subunit REV1 and on the presence of RNA (Aim 1.2). The second part of my research will elucidate RT-DSBR activity via an mRNA donor transcribed at the site of the break (Aim 2). I will detect if RT-DSBR occurs endogenously at highly transcribed genes by using intron loss as a proxy for the use of a spliced mRNA repair template (Aim 1.2). Finally, I will validate CRISPR-Cas9 screen hits via the intron loss assay to determine which proteins are involved in various steps of the RT-DSBR pathway (Aim 2.2). Elucidating new roles for RNA and reverse transcription in DSB repair would expand the fields of genome integrity and could lead to potential new targets for cancer therapy. Since RT-DSBR would be a more accurate repair pathway in post-mitotic cells that only rely on the error-prone NHEJ, this research could also elucidate novel DNA repair in cells like neurons. Project Number: 1F31CA305886-01 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Hina Shah | Institution: WEILL MEDICAL COLL OF CORNELL UNIV, NEW YORK, NY | Award Amount: $49,538 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F08-L (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11240481