Molecular Mechanisms of DNA Interstrand Cross-Link Repair by the Fanconi Anemia Pathway

Author: Altshuller, Maria

Year: 2026

Degree: Dissertation (Ph.D.)

Advisor: Semlow, Daniel R.

Committee Members: Shan, Shu-ou; Dunphy, William G.; Guttman, Mitchell; Voorhees, Rebecca M.; Semlow, Daniel R.

Option: Biochemistry and Molecular Biophysics

Abstract

DNA interstrand cross-links (ICLs), which consist of a covalent link between complementary DNA strands, are among the most cytotoxic lesions that cells encounter, as the ICL blocks strand separation during replication and transcription. The Fanconi anemia (FA) pathway is the primary mechanism through which proliferating cells repair ICLs encountered during DNA replication, and mutations in FA pathway genes cause the inherited bone marrow failure and cancer predisposition syndrome Fanconi anemia. Despite considerable progress in elucidating the molecular mechanism of FA pathway-mediated ICL repair, fundamental questions remain about how specific ICL substrates are processed and how the replication fork dynamics that accompany repair are regulated. This thesis addresses these questions using cell-free extracts prepared from Xenopus laevis eggs, which enable biochemical characterization of ICL repair. We first investigated the repair of ICLs induced by colibactin, a gut microbiome-derived genotoxin implicated in the pathogenesis of colorectal cancer. We demonstrate that the FA pathway is responsible for replication-coupled repair of colibactin-induced ICLs and identify the translesion synthesis polymerases that bypass the unhooked ICL remnant, providing mechanistic insight into how cells process this clinically relevant lesion. We then uncovered a phosphoregulatory axis governing replication fork dynamics during ICL repair, showing that the ataxia-telangiectasia mutated (ATM) kinase promotes nucleolytic resection of reversed fork intermediates through the nucleases EXO1 and DNA2-WRN, and that this resection is constrained by opposing PP2A phosphatase activity. This regulatory mechanism ensures productive fork restoration. Finally, we present preliminary evidence that X-shaped replication intermediates are sufficient to activate FA pathway signaling and ICL unhooking in the absence of ongoing replication, suggesting that these structures may serve as the trigger for FA pathway activation independently of the replisome. Taken together, this work advances our mechanistic understanding of how cells tolerate ICL stress and illuminates regulatory mechanisms that ensure accurate and efficient ICL repair.