Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA.
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA.
J Biol Chem. 2021 Sep;297(3):101025. doi: 10.1016/j.jbc.2021.101025. Epub 2021 Jul 30.
The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein-protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER.
碱基切除修复 (BER) 途径涉及 DNA 聚合酶 (pol) β 填补缺口，随后由 ligase IIIα 封闭缺口。X 射线交叉互补蛋白 1 (XRCC1) 是一种非酶类支架蛋白，可组装多蛋白复合物，尽管 XRCC1 协调协调 BER 的最后步骤的机制尚未完全定义。在这里，我们使用生化和生物物理方法的组合，揭示了 polβ/XRCC1 复合物在正确核苷酸插入 DNA 中的缺口后增加了 BER 反应的连续性，并增强了带有缺口的修复产物向最终连接步骤的交接。此外，在 XRCC1 存在下，polβ 8-氧鸟苷三磷酸插入后带有缺口的修复中间产物的诱变连接得到增强。我们的结果表明 XRCC1 对 polβ/dNTP/缺口 DNA 和 ligase IIIα/ATP/缺口 DNA 催化三元复合物的形成具有稳定作用。蛋白质-蛋白质相互作用和 DNA 结合动力学的实时监测表明，XRCC1 与 polβ 的结合比与 ligase IIIα 或 aprataxin 的结合更强，并且对带有未受损或受损末端的缺口 DNA 的亲和力比对一个核苷酸缺口修复中间体的亲和力更高。最后，我们证明了由于癌症相关（P161L、R194W、R280H、R399Q、Y576S）和小脑共济失调相关（K431N）XRCC1 变体，稳定的 polβ/XRCC1 复合物形成、polβ 和 ligase IIIα 蛋白相互作用动力学以及交接过程存在细微差异。总体而言，我们的研究结果为 XRCC1 的协调作用以及其疾病相关变体对多蛋白/DNA 复合物中有效 BER 的底物产物通道化的影响提供了新的见解。
