The FHA domain of PNKP is essential for its recruitment to DNA damage sites and maintenance of genome stability
The FHA domain of PNKP is essential for its recruitment to DNA damage sites and maintenance of genome stability
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DOI：
10.1016/j.mrfmmm.2020.111727
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发表时间：
2021-01-01
影响因子：
2.3
通讯作者：
Matsumoto, Yoshihisa
中科院分区：
文献类型：
--
作者：
Tsukada, Kaima;Shimada, Mikio;Matsumoto, Yoshihisa
多核苷酸激酶磷酸酶（PNKP）具有DNA末端激酶和磷酸酶的双重酶活性，是连接的先决条件，参与碱基切除修复、单链断裂修复和非同源末端连接的双链断裂修复。在这项研究中，我们研究的招聘机制PNKP DNA损伤位点的激光显微照射和活细胞成像分析，共聚焦显微镜。我们发现，PNKP的叉头相关（FHA）结构域是必不可少的招聘PNKP的DNA损伤位点。FHA结构域中的Arg 35和Arg 48是与XRCC 1和XRCC 4相互作用所必需的。PNKP R35 A/R48 A突变体不能在激光径迹上积累，siRNA介导的XRCC 1和/或XRCC 4的缺失减少了PNKP在激光径迹上的积累，表明PNKP通过其FHA结构域与XRCC 1或XRCC 4之间的相互作用被募集到DNA损伤位点。此外，表达PNKP R35 A/R48 A突变体的细胞表现出对电离辐射的敏感性增加，与延迟SSB和DSB修复和基因组不稳定性相关，以微核和染色体桥为代表。总之，这些发现揭示了PNKP通过其FHA结构域募集到DNA损伤位点对于DNA修复和维持基因组稳定性的重要性。
Polynucleotide kinase phosphatase (PNKP) has dual enzymatic activities as kinase and phosphatase for DNA ends, which are the prerequisite for the ligation, and thus is involved in base excision repair, single-strand break repair and non-homologous end joining for double-strand break (DSB) repair. In this study, we examined mechanisms for the recruitment of PNKP to DNA damage sites by laser micro-irradiation and live-cell imaging analysis using confocal microscope. We show that the forkhead-associated (FHA) domain of PNKP is essential for the recruitment of PNKP to DNA damage sites. Arg35 and Arg48 within the FHA domain are required for interactions with XRCC1 and XRCC4. PNKP R35A/R48A mutant failed to accumulate on the laser track and siRNAmediated depletion of XRCC1 and/or XRCC4 reduced PNKP accumulation on the laser track, indicating that PNKP is recruited to DNA damage sites via the interactions between its FHA domain and XRCC1 or XRCC4. Furthermore, cells expressing PNKP R35A/R48A mutant exhibited increased sensitivity toward ionizing radiation in association with delayed SSB and DSB repair and genome instability, represented by micronuclei and chromosome bridges. Taken together, these findings revealed the importance of PNKP recruitment to DNA damage sites via its FHA domain for DNA repair and maintenance of genome stability.