NMR STRUCTURAL AND BIOPHYSICAL STUDIES OF XRCC1

描述：(改编自申请人的摘要)X光交叉互补 蛋白1(XRCC1)参与DNA损伤的修复，在小鼠中， 也被认为是胚胎发育所必需的。XRCC1相互作用 与DNA修复蛋白多聚腺苷二磷酸核糖聚合酶(PARP)、DNA聚合酶 (β-Pol)和DNA连接酶III。XRCC1、β-Pol和DNA连接酶形成的复合体 DNA连接酶III修复在最后三步切割的基本部位 碱基切除修复途径。XRCC1的N-末端结构域已被证明 与单链断裂DNA的特异性结合及其结构最近 用高分辨核磁共振方法进行了测定。这项建议旨在 表征N-末端相互作用的分子机制 具有DNA底物和β-POL的XRCC1结构域。诱变方法，在 结合生化分析，将用于阐明这些残留物 这有助于约束。现有的基于核磁共振的结构和化学位移 结合表面的微扰图将指导诱变。完整的 还将探索DNA底物专一性的范围。主干动力学 将被表征并与功能结合位点相关联。40 kDa 复合体，涉及XRCC1的N-末端结构域、DNA底物和β-Pol， 将使用基于核磁共振的方法对其结构进行表征。

DESCRIPTION: (adapted from the applicant's abstract) Xray cross complementing protein 1 (XRCC1) participates in the repair of DNA damage and, in mice, has been indicated to also be required for embryonic development. XRCC1 interacts with the DNA repair proteins poly-ADP-ribose polymerase (PARP), DNA polymerase (beta-Pol), and DNA ligase III. The complex formed between XRCC1, beta-Pol and DNA ligase III repairs incised abasic sites in the final three steps of the base excision repair pathway. The N-terminal domain of XRCC1 has been shown to bind specifically to single-strand break DNA and its structure has recently been determined by high resolution NMR methods. This proposal seeks to characterize the molecular mechanism of the interaction of the N-terminal domain of XRCC1 with DNA substrate and with beta-Pol. Mutagenesis methods, in combination with biochemical assays, will be used to elucidate those residues that contribute to binding. The existing NMR-based structure and chemical shift perturbation map of the binding surface will guide the mutagenesis. The full range of DNA substrate specificity will also be explored. Backbone dynamics will be characterized and correlated with functional binding sites. The 40 kDa complex, involving the N-terminal domain of XRCC1, DNA substrate and beta-Pol, will be structurally characterized using NMR-based methods.