Crystal Structures of Human Exonuclease I with DNA Provides Insight into Substrate Selectivity and Mechanism

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Human Exonuclease 1 (hEXO1) plays an essential role in the maintenance of genomic stability through nuclease activity on DNA intermediates involved in replication, recombination, and repair. hEXO1 is a member of the structure-specific 5’ nuclease family, which includes FEN-1 (flap endonuclease 1), GEN-1 (gap endonulcease 1), and XPG (xeroderma pigmentosum complementation group G). This family possesses highly divergent protein sequences and DNA substrate specificities. We present crystal structures of the hEXOI catalytic domain in complex with a 5’ recessed DNA substrate, which represents an intermediate structure in mismatch repair. The structure contains a core region seen in a variety of homologous FEN-1 structures; however, certain nucleotide binding motifs are altered or repositioned. These structures with DNA bound in an assembled active site provide the insight into the mechanism of EXO1 and important family members. Contacts to the DNA are made primarily to the complementary strand, anchored by a potassium-binding motif observed previously in Pol β. Additional contacts are made through a hydrophobic wedge which induces a ∼90° bend in the DNA. The 5’ substrate strand is held in place mainly by base pairing; however, two bases must unpair from the substrate strand to reach the active site. In a product complex the terminal base in the active site is flipped and stabilized by pi-stacking interactions with a tyrosine. The terminal phosphate is coordinated by two Mn2+ ions in a geometry consistent with other nucleases that cleave DNA using a two-metal ion mechanism for catalysis. Structures of hEXO1 will greatly enhance understanding of substrate selection mechanisms and nuclease family mode of action, as well as allowing us to model additional substrates. Analysis of these structures may permit identification of protein-interaction surfaces critical to DNA repair.