EEPD1 Evolved a Unique DNA Clamping Dimer Protecting Reversed Replication Forks

Runze Shen (Advisor: John Tainer, PhD)

           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 structure, assembly, and function at stalled DNA replication fork intermediates. Combined results show that 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. X-ray scattering, 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 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.

Advisory Committee:

• John Tainer, PhD, Chair
• Junjie Chen, PhD
• Susan Rosenberg, PhD
• Irina Serysheva, PhD
• Richard Wood, PhD

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