Genomics Research Center, Academia Sinica, Taipei, Taiwan.
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
FASEB J. 2019 Feb;33(2):2017-2025. doi: 10.1096/fj.201800856R. Epub 2018 Sep 10.
Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.
脱氧胸苷三磷酸（dTTP）的细胞供应对 DNA 复制和修复至关重要。胸苷激酶（TMPK）催化胸苷单磷酸转化为胸苷二磷酸，这是 dTTP 合成的必要步骤。尽管 TMPK 和核糖核苷酸还原酶（RNR）主要在细胞质中定位，但它们在 DNA 损伤部位被检测到，用于局部 dNDP 的形成。因为脱氧尿苷二磷酸是由 RNR 合成的，因此 TMPK 和 RNR 同时被招募到 DNA 损伤部位对于防止脱氧尿苷三磷酸介导的毒性修复至关重要。本研究调查了 TMPK 被招募到 DNA 损伤部位的机制。我们的数据表明，共济失调毛细血管扩张突变（ATM）激酶活性是 TMPK 被招募到 DNA 损伤部位所必需的。此外，我们发现 TMPK 能够与组蛋白乙酰转移酶 Tip60 和 RNR 形成复合物。ATM 激酶抑制剂降低了复合物的形成和 TMPK 的磷酸化。我们的分析进一步表明 TMPK 在丝氨酸 88 处发生磷酸化，这是 ATM 激酶的一个保守位点。该位点的磷酸化缺陷突变抑制了 TMPK 向 DNA 损伤部位的募集以及与 Tip60 的复合物形成。最后，我们提供了证据表明该位点对于 TMPK 在 DNA 修复中的功能至关重要，但对于催化活性并非如此。综上所述，这些发现表明 Tip60-ATM 信号转导对 TMPK 向 DNA 损伤部位的募集有功能贡献，从而增加了用于 DNA 修复的局部 dTTP 合成。
