| 摘要: | Endonuclease G（EndoG）是真核生物中演化上共同保留下來的內切酶，主要位於粒線體内。目前研究已經提出EndoG可能參與粒線體DNA（mtDNA）複製、在氧化壓力下維持mtDNA完整性，並在細胞凋亡和早期胚胎發育期間降解细胞核DNA。然而，目前仍不清楚EndoG作用的核酸受質是什麼，以及EndoG如何優先切割這些核酸。為了回答這些問題，首先我們的研究重點是探討人類EndoG（hEndoG）其在結合和切割各種核酸受質方面的偏好，包括單股DNA（ssDNA）、雙股DNA（dsDNA）、有缺口的dsDNA(gapped dsDNA)、有切口的dsDNA(nicked dsDNA)、攜帶氧化鳥嘌呤的dsDNA（oxoG-DNA）和5-羥甲基胞嘧啶修飾的dsDNA（5hmC-DNA），以及單股RNA（ssRNA）和RNA/DNA混合雙股。我們的研究结果發現，hEndoG對ssDNA、ssRNA、dsDNA和有修飾的dsDNA的結合親和力高出RNA/DNA混合雙股約10倍。此外，hEndoG偏好切割受到氧化損傷的DNA，包括gapped dsDNA, nicked dsDNA和oxoG-DNA。在降解有缺口的和帶切口的dsDNA時，hEndoG優先在帶有缺口或切口位點的對面股上進行切割，而在降解oxoG-DNA和5hmC-DNA時，則優先在攜帶有修飾鹼基股的對面股進行切割。hEndoG的晶體結構顯示出二聚體構象，具有一對His-Me finger motif用於核酸結合和切割。我們建構了hEndoG與ssDNA和有gapped dsDNA结合的結構模型，顯示它偏好切割ssDNA和gapped dsDNA，可能是因為這類核酸分子較易彎曲，導致且其切割位點的磷酸分子與位於His-Me finger motif中的Mg2+離子距離較近，較易進行水解反應。總結來說，我們的結果支持hEndoG在氧化壓力條件下，擔任切割並去除氧化傷害的DNA，扮演著維護粒線體基因完整性的關鍵角色。;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. |
