Department of Oncology, University of Alberta and Experimental Oncology, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada, T6G 1Z2.
Nucleic Acids Res. 2012 Apr;40(8):3484-95. doi: 10.1093/nar/gkr1245. Epub 2011 Dec 30.
Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly examined. Polynucleotide kinase/phosphatase (PNKP) processes strand-break termini to render them chemically compatible for the subsequent action of DNA polymerases and ligases. Here, we demonstrate that functionally active full-length PNKP is present in mitochondria as well as nuclei. Downregulation of PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H(2)O(2)-induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria.
线粒体 DNA（mtDNA）中的突变与广泛的人类疾病和衰老有关。与核 DNA 相比，由于 mtDNA 靠近呼吸链，并且缺乏染色质相关蛋白的保护，因此更容易受到氧化损伤。虽然已经对通过碱基切除修复途径修复 mtDNA 中碱基的氧化损伤进行了深入研究，但对 mtDNA 中氧化诱导的链断裂的修复研究还不够彻底。多核苷酸激酶/磷酸酶（PNKP）处理链断裂末端，使其在化学上适合随后的 DNA 聚合酶和连接酶的作用。在这里，我们证明功能性全长 PNKP 存在于线粒体和细胞核中。PNKP 的下调导致过氧化氢处理细胞中线粒体 DNA 中链断裂的积累。完全恢复 H（2）O（2）诱导的线粒体中链断裂的修复需要 PNKP 的激酶和磷酸酶活性。我们还证明 PNKP 在线粒体蛋白 mitofilin 附近含有一个线粒体靶向信号。我们进一步表明 PNKP 与线粒体蛋白 mitofilin 相关联。与 mitofilin 的相互作用可能有助于将 PNKP 转运到线粒体中。
