| dc.description.abstract | Endonuclease G (EndoG) is an evolutionarily conserved endonuclease in eukaryotes primarily located in mitochondria. EndoG has been suggested to involve in mitochondrial DNA (mtDNA) replication, maintaining mtDNA integrity under oxidative stress and degrading nuclear DNA during apoptosis and early embryogenesis. However, it remains unclear what are the EndoG-targeting nucleic acids substrates, and how could EndoG preferentially cleave these nucleic acids. To address these questions, our study focused on investigating human EndoG (hEndoG) to reveal its preferences in binding and cleaving various nucleic acid substrates, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), nicked dsDNA, gapped dsDNA, modified dsDNA harboring oxidized guanine (oxoG-DNA) and hydroxymethylated cytosine (5hmC-DNA), as well as single-stranded RNA (ssRNA) and RNA/DNA hybrid duplexes. Our findings reveal that hEndoG demonstrates a ~10-fold higher binding affinities for ssDNA, ssRNA, dsDNA, and modified dsDNA over RNA/DNA hybrid duplexes. Moreover, hEndoG exhibits a preference for cleaving oxidatively damaged DNA, including gapped dsDNA, nicked dsDNA, and oxoG-DNA. In degrading gapped and nicked DNA, hEndoG preferentially cleaves at the opposite strand of the gapped/nicked site, whereas in degrading oxoG-DNA and 5hmC-DNA, it preferentially cleaves at the opposite strand of the one harboring the modified base. The crystal structure of hEndoG was determined revealing a dimeric conformation with a pair of His-Me finger motifs for nucleic acid binding and cleavage. We constructed structural models of hEndoG bound with ssDNA and gapped DNA, showing that it prefers to cleave ssDNA and gapped DNA likely because these nucleic acids could adopt kinked DNA structures with a scissile phosphate located closely to the catalytic Mg2+ ion in the His-Me finger motif. Overall, our results strongly support the notion that hEndoG plays a critical role in safeguarding mitochondrial genome integrity under oxidative stress conditions by targeting and removing oxidatively damaged DNA. | en_US |