Dissertations and Theses (Open Access)

Author ORCID Identifier

0000-0003-0892-9845

Date of Graduation

5-2025

Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

John A. Tainer

Committee Member

Richard D. Wood

Committee Member

Junjie Chen

Committee Member

Susan M. Rosenberg

Committee Member

Irina I. Serysheva

Abstract

Exonuclease/endonuclease/phosphatase (EEP)-fold hydrolases are canonically monomeric phosphodiesterases exemplified by APE1, DNase I, and TDP2 nucleases. While EEP superfamily domain-containing protein 1 (EEPD1) acts in DNA stress responses, its proposed nuclease activities are enigmatic. Here, we integrate hybrid structural methods, evolution, biochemistry, cancer genomics, molecular and cell biology to define EEPD1’s structure, assembly, and function at stalled DNA replication fork intermediates. Combined results show that EEPD1 surprisingly requires both the unique EEP domain dimer and distinctive tandem Helix-hairpin-Helix [(HhH)2] domains to clamp double-stranded(ds) DNA at reversed DNA replication forks for fork protection. X-ray scattering, crystal, and cryo-EM structures unveil an unprecedented tryptophan-handshake dimer, a conserved interface di-Trp-Pro pocket, and an adjustable "wrist" enabling an open-closed conformational switch. EEPD1 dimer preferentially binds complex dsDNA replication fork intermediates, but alone lacks nuclease activity due to its loss of key EEP catalytic residues during Metazoan evolution and atmospheric oxygen buildup. Instead, EEPD1 prevents nucleolytic degradation of reversed replication forks by MRE11. Consistently, cancer bioinformatics support oxidative damage-dependent EEPD1 association as a significant modulator of overall patient survival. Collective findings uncover unexpected EEP dimer and fork protection function in clamping, not cutting, reversed replication forks for metazoan oxidative stress responses controlling genome stability and cancer outcomes.

Keywords

EEPD1, structrual biology, replication stress

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Available for download on Wednesday, November 04, 2026

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