# EEPD1 evolved a unique DNA clamping dimer protecting reversed replication forks

## TL;DR

This study reveals that EEPD1 protects DNA replication forks by clamping them, not cutting them, which helps maintain genome stability and affects cancer outcomes.

## Contribution

The paper discovers EEPD1's unique DNA clamping dimer function in protecting reversed replication forks, challenging its proposed nuclease role.

## Key findings

- EEPD1 uses a dimeric EEP domain and (HhH)2 domains to clamp DNA at reversed replication forks.
- EEPD1 lacks nuclease activity due to loss of catalytic residues during evolution but prevents MRE11-mediated degradation.
- Cancer data show EEPD1's role in oxidative stress responses correlates with patient survival.

## Abstract

Exonuclease/endonuclease/phosphatase (EEP)-fold hydrolases are canonically monomeric phosphodiesterases exemplified by APE1, DNase I, and TDP2 nucleases. While EEP family 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, plus molecular and cell biology to define EEPD1 structure, assembly, and function at stalled DNA replication forks. Results imply EEPD1 surprisingly requires both 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. Small-angle X-ray Scattering (SAXS), crystal, and cryo-EM structures unveil an unprecedented tryptophan handshake dimer, conserved interface di-Trp-Pro pocket, and adjustable “wrist” enabling an open-closed conformational switch. EEPD1 dimer cooperatively binds complex dsDNA replication fork intermediates but alone lacks nuclease activity due to loss of key EEP catalytic residues during Metazoan evolution and atmospheric oxygen buildup. Instead, EEPD1 prevents nucleolytic degradation of reversed replication forks by MRE11. Furthermore, 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 cleaving, reversed replication forks for metazoan oxidative stress responses controlling genome stability and cancer outcomes.

Graphical Abstract