Function and Regulation of TRAIP at Replisome-Blocking DNA Lesions - Project Summary/Abstract
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.
