Elucidating the Mitochondrial and Nuclear functions of ATP Synthase Subunit ATP5A1 that Maintain Genome Integrity in Response to Oxidative Stress

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PROJECT SUMMARY – Maintenance of genome integrity is a fundamental function of all cellular life. Respiring organisms maintain robust antioxidant systems to defend against pathologic reactive oxygen species (ROS) which are a threat to genome integrity. ROS activates the DNA damage response (DDR) by virtue of several types of DNA damage including crosslinks between RNA and DNA as well as induction of DNA double stranded breaks (DSBs). The most common cancer therapeutic that mediates cell kill through induction of ROS is ionizing radiation (IR). However, the mechanisms by which the DDR responds to ROS to maintain genome integrity are not well understood. The PI, Neil Pfister, MD, PhD, identified ATP5A1 as a top hit in a genome-wide CRISPR/Cas9 knockout screen using IR as the selective pressure. ATP5A1 is the alpha subunit of the soluble F1 subunit of ATP synthase, the terminal electron transport chain complex that generates ATP in the presence of an electrochemical proton gradient and molecular oxygen. A cleaved form of ATP5A1 was found to co-localize to poly(ADP-ribose) and γH2AX foci, which is enhanced by oxidative stress and inhibited by PARP inhibition. Cleaved ATP5A1 contains a poly(ADP-ribose) interaction domain that is required for poly(ADP-ribose) binding and localization to DSBs. R-loop resolution proteins DHX9 and hnRNPU were identified as top protein interactors, and depletion of ATP5A1, DHX9, or hnRNPU significantly increased levels of R-loops and spontaneous DSBs. This project examines the central hypothesis that cleaved ATP5A1 cooperates with poly(ADP-ribose) polymerases to facilitate R-loop resolution and genome maintenance in response to oxidative stress. To test this hypothesis, 3 specific aims are proposed. Specific Aim 1 will determine how cleaved ATP5A1 is regulated. Specific Aim 2 will delineate how cleaved ATP5A1 promotes genome maintenance through interaction with DHX9, hnRNPU, and poly(ADP-ribose). Specific Aim 3 will dissect how cleaved ATP5A1 impacts cell fate following oxidative stress. Dr. Pfister is mentored by Dr. David Yu and Dr. Kathy Griendling with additional support from Dr. Francesca Storici, Dr. Xingming Deng, and Dr. William Dynan. Emory University boasts an outstanding research environment at an NCI-designated Comprehensive Cancer Center to complete the proposed research. The goal of the K08 career development award is for Dr. Pfister to receive career mentorship and training in genome maintenance and cell fate, ROS and oxidative metabolism, mitochondrial biology, and R-loop biology, which complements his past training in order to investigate the role of cleaved ATP5A1 in coordinating oxidative metabolism with genome maintenance, a topic of critical importance to cancer initiation and cancer treatment. The proposed research, in combination with a structured mentoring and training plan, is designed to facilitate Dr. Pfister's long-term goal to supervise an independently funded laboratory that investigates how cells respond to IR and ROS in order to identify new opportunities for cancer therapy.