Department of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Section of Computational and Molecular Biology, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, 1441 Eastlake Ave, Rm. 5428, Los Angeles, CA 90089, USA.
Department of Internal Medicine and Molecular Genetics and Microbiology, and the University of New Mexico Comprehensive Cancer Center, University of New Mexico, 915 Camino de Salud, Albuquerque, NM 87131, USA.
Nucleic Acids Res. 2020 Dec 16;48(22):12746-12750. doi: 10.1093/nar/gkaa1118.
As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.
作为核苷酸转移酶，形成共价酶-腺苷酸中间物是所有 DNA 连接酶的共同第一步。虽然已经表明真核 DNA 连接酶利用 ATP 作为腺苷酸化供体，但最近有报道称，人 DNA 连接酶 IV 也可以利用 NAD+，并且在较小程度上利用 ADP-核糖作为腺苷酸基团的来源，并且 NAD+不同于 ATP，通过支持多个催化循环来增强连接。由于这一意外发现对我们理解 DNA 双链断裂修复的机制和调控具有重要意义，我们试图证实 NAD+和 ADP-核糖可以被人 DNA 连接酶 IV 用作辅助因子。在这里，我们提供的证据表明 NAD+不会通过预腺苷酸化的 DNA 连接酶 IV 增强连接，表明该辅助因子不用于再腺苷酸化和随后的连接循环。此外，我们发现去腺苷酸化的 DNA 连接酶 IV 的连接依赖于 ATP 而不是 NAD+或 ADP-核糖。因此，我们得出结论，人 DNA 连接酶 IV 不能将 NAD+或 ADP-核糖用作连接的腺苷酸化供体。
