Each time a cell divides, its genome must be accurately and rapidly duplicated. However, DNA replication can be blocked by diverse types of DNA damage. DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs) are especially formidable challenges to replication because they impede the progression of the replicative CDC45/MCM2-7/GINS (CMG) helicase. Failure to repair these lesions can lead to genomic instability and cancer development. ICLs and DPCs are primarily sensed and repaired during S phase when replication forks stall at the lesion. Using Xenopus egg extracts, we have reconstituted replication-coupled ICL and DPC repair. In this system, ICL repair is initiated by the convergence of two replication forks at the lesion. Fork convergence triggers polyubiquitylation of the CMGs, activating one of two ICL repair mechanisms ? the NEIL3 pathway or the Fanconi anemia (FA) pathway. Similarly, DPC repair is initiated after replisome collision triggers DPC ubiquitylation, marking the crosslinked protein for proteolytic degradation by the proteasome and the protease SPRTN. Thus, ubiquitin signaling plays a key role in regulating ICL and DPC repair. We have recently identified TRAIP as the E3 ubiquitin ligase responsible for ubiquitylation of CMGs stalled at ICLs and of DPCs. How TRAIP is regulated to act in these diverse situations remains unclear. We have found that TRAIP is constitutively assembled with the replisome, positioned at its leading edge. This would allow TRAIP to ubiquitylate any proteinaceous barrier encountered by the replisome, including DPCs and abutting replisomes at an ICL. The proposed studies seek to understand the mechanisms and regulation of TRAIP in these contexts.
Aim 1 addresses how TRAIP ubiquitylates replisome barriers while avoiding premature replisome disassembly using a combination of biochemical reconstitution, single-molecule imaging, and structural analysis.
Aim 2 investigates whether also TRAIP functions independently of the replisome and uses unbiased proteomic and functional genomic approaches to identify novel TRAIP regulators and effectors. I will perform the mentored phase of this work at Harvard Medical School (HMS) under the combined mentorship of Dr. Johannes Walter and an assembled advisory committee of expert scientists in diverse fields. During this training period, I will add to my previous biochemical experience, gaining skills in cellular assays to investigate DNA damage and repair, as well as learning cryo-EM, mass spectrometry, and CRISPR screening techniques. This mentorship, along with the dynamic research environment and abundant career development resources at HMS, will help me realize my goal of leading an independent research program, where I will work toward understanding the cell?s response to DNA damage and the mechanisms of faithful genome duplication. The proposed research will deepen our understanding of how replisomes handle fork-stalling barriers and will shed light on how cancer cells respond to chemotherapy-induced lesions. These insights may lead to new ways of treating DNA repair deficiency disorders and to new strategies to potentiate or reactivate cytotoxic cancer chemotherapeutics.

Public Health Relevance

DNA replication, the fundamental cellular process by which our genomes are propagated, can be blocked by various forms of DNA damage including DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs). We have recently uncovered a key role for the enzyme TRAIP in ICL and DPC repair. Our studies will delineate the mechanisms of TRAIP function and regulation, deepening our understanding of how cells repair these toxic lesions to avoid cancer and how cancer cells respond to chemotherapy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Career Transition Award (K99)
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Special Emphasis Panel (ZGM1)
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Gaillard, Shawn R
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Harvard Medical School
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