Aceytuno R Daniel, Piett Cortt G, Havali-Shahriari Zahra, Edwards Ross A, Rey Martial, Ye Ruiqiong, Javed Fatima, Fang Shujuan, Mani Rajam, Weinfeld Michael, Hammel Michal, Tainer John A, Schriemer David C, Lees-Miller Susan P, Glover J N Mark
Department of Biochemistry, University of Alberta, Edmonton, AB T6G-2H7, Canada.
Department of Biochemistry & Molecular Biology, Robson DNA Science Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, AB T2N 1N4, Canada.
Nucleic Acids Res. 2017 Jun 2;45(10):6238-6251. doi: 10.1093/nar/gkx275.
Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.
非同源末端连接（NHEJ）修复非循环真核细胞中的DNA双链断裂。NHEJ依赖于多核苷酸激酶/磷酸酶（PNKP），它产生5΄-磷酸/3΄-羟基DNA末端，这对于NHEJ DNA连接酶LigIV的连接至关重要。PNKP和LigIV需要NHEJ支架蛋白XRCC4。PNKP的FHA结构域与CK2磷酸化的XRCC4 C末端尾巴结合，而LigIV利用其串联的BRCT重复序列结合XRCC4的卷曲螺旋。然而，组装好的PNKP-XRCC4-LigIV复合物仍未得到表征。在这里，我们报告了重组PNKP-XRCC4-LigIV复合物的纯化和表征。我们表明，该复合物中PNKP的稳定结合需要XRCC4磷酸化，并且每个XRCC4二聚体仅结合一个PNKP原聚体。小角X射线散射（SAXS）揭示了一种灵活的多态复合物，这表明PNKP FHA和催化结构域均与XRCC4卷曲螺旋和LigIV BRCT重复序列接触。氢-氘交换表明PNKP磷酸酶结构域上可能与XRCC4-LigIV接触的一个表面受到保护。该表面上的一个突变（E326K）导致遗传性神经发育障碍MCSZ。这种突变损害了PNKP在人类细胞中募集到受损DNA的能力，并提供了一种可能的疾病机制。总之，这项工作揭示了PNKP与XRCC4-LigIV之间的多点接触，这些接触调节了NHEJ中PNKP的募集和活性。
