Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065.
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032.
Proc Natl Acad Sci U S A. 2021 Mar 16;118(11). doi: 10.1073/pnas.2023955118.
Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3' to 5' translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet-like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB-DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.
分枝杆菌 AdnAB 是一种异源二聚体解旋酶-核酸酶，通过切除 DNA 双链断裂（DSB）来启动同源重组。AdnB 亚基的 N 端马达结构域水解 ATP，以驱动 AdnAB 在追踪 DNA 链上快速和连续的 3'到 5'易位。当与 ATP 酶循环同步的蛋白质结构域运动的振荡将 DNA 追踪链向前推进一个核苷酸步时，ATP 水解具有机械生产力，这被认为需要爪和棘轮式的方式。通过测量 AdnB-DNA 界面上 16 个氨基酸的丙氨酸突变对依赖 DNA 的 ATP 水解、DNA 易位和 DSB 切除的整体和单分子测定的影响，我们深入了解了哪些 DNA 接触将 ATP 水解与马达活性偶联。结果表明，AdnB 的色氨酸 325 插入追踪链并堆积在核碱基上，是棘轮爪的单一必需成分，没有它，ssDNA 上的 ATP 水解在机械上是徒劳的。 Thr663 和 Thr118 与追踪链磷酸的接触以及 His665 与核碱基的接触丧失，会在 DSB 切除过程中使 AdnAB 马达急剧减速。我们对 AdnAB 的发现促使我们将其机制类比为汽车离合器。
