Mizushina Y, Ohkubo T, Sugawara F, Sakaguchi K
Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Biochemistry. 2000 Oct 17;39(41):12606-13. doi: 10.1021/bi001276m.
The purpose of this study was to investigate the molecular action of lithocholic acid (LCA), known as a selective inhibitor of DNA polymerase beta (pol beta). The 39-kDa pol beta was separated proteolytically into two fragments of the template-primer binding domain (8 kDa) and the catalytic domain (31 kDa). LCA bound tightly to the 8-kDa fragment but not to the 31-kDa fragment. We examined the structural interaction with the 8-kDa domain using LCA. On (1)H-(15)N HMQC NMR analysis of pol beta with LCA, the 8-kDa domain bound to LCA as a 1:1 complex with a dissociation constant (K(D)) of 1.56 mM. The chemical shifts were observed only in residues mainly in helix-3, helix-4, and the 79-87 turn of the same face. No significant shifts were observed for helix-1, helix-2, and other loops of the 8-kDa domain. This region was composed mainly of three amino acid residues (Lys60, Leu77, and Thr79) of pol beta on the LCA interaction interface. The inhibition mechanism and the structure-function relationship between pol beta and LCA is discussed.
本研究的目的是探究石胆酸（LCA）的分子作用，已知其为DNA聚合酶β（polβ）的选择性抑制剂。39 kDa的polβ经蛋白酶解分离为模板 - 引物结合结构域（8 kDa）和催化结构域（31 kDa）的两个片段。LCA紧密结合于8 kDa片段，但不与31 kDa片段结合。我们使用LCA研究了与8 kDa结构域的结构相互作用。对与LCA结合的polβ进行（1）H - （15）N HMQC NMR分析时，8 kDa结构域与LCA以1:1复合物形式结合，解离常数（K（D））为1.56 mM。化学位移仅在主要位于同一面的螺旋 - 3、螺旋 - 4以及79 - 87转角处的残基中观察到。对于8 kDa结构域的螺旋 - 1、螺旋 - 2和其他环，未观察到明显的位移。该区域主要由LCA相互作用界面上polβ的三个氨基酸残基（Lys60、Leu77和Thr79）组成。讨论了polβ与LCA之间的抑制机制以及结构 - 功能关系。
