Maciejewski M W, Liu D, Prasad R, Wilson S H, Mullen G P
Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA.
J Mol Biol. 2000 Feb 11;296(1):229-53. doi: 10.1006/jmbi.1999.3455.
Mammalian DNA polymerase beta functions in the base excision DNA repair pathway filling in short patches (1-5 nt) in damaged DNA and removing deoxyribose 5'-phosphate from the 5'-side of damaged DNA. The backbone dynamics and the refined solution structure of the N-terminal domain of beta-Pol have been characterized in order to establish the potential contribution(s) of backbone motion to the DNA binding and deoxyribose 5'-phosphate lyase function of this domain. The N-terminal domain is formed from four helices packed as two antiparallel pairs with a 60 degrees crossing between the pairs. The RMSD of the NMR conformers (residues 13-80) is 0.37 A for the backbone heavy atoms and 0.78 A for all heavy atoms. NMR characterization of the binding site(s) for a ssDNA-5mer, ssDNA-8mer, ssDNA-9mer, and dsDNA-12mer shows a consensus surface for the binding of these various DNA oligomers, that surrounds and includes the deoxyribose 5'-phosphate lyase active site region. Connection segments between helices 1 and 2 and between helices 3 and 4 each contribute to DNA binding. Helix-3-turn-helix-4 forms a helix-hairpin-helix motif. The highly conserved hairpin sequence (LPGVG) displays a significant degree of picosecond time-scale motion within the backbone, that is possibly important for DNA binding at the phosphodiester backbone. An Omega-loop connecting helices 1 and 2 and helix-2 itself display significant exchange contributions (R(ex)) at the backbone amides due to apparent conformational type motion on a millisecond time-scale. This motion is likely important in allowing the Omega-loop and helix-2 to shift toward, and productively interact with, gapped DNA. The deoxyribose 5'-phosphate lyase catalytic residues that include K72 which forms the Schiff's base, Y39 which is postulated to promote proton transfer to the aldehyde, and K35 which assists in phosphate elimination, show highly restricted backbone motion. H34, which apparently participates in detection of the abasic site hole and assists in the opening of the hemiacetal, shows conformational exchange.
哺乳动物DNA聚合酶β在碱基切除DNA修复途径中发挥作用，填补受损DNA中的短片段（1 - 5个核苷酸），并从受损DNA的5'端去除脱氧核糖5'-磷酸。对β-聚合酶N端结构域的主链动力学和精细溶液结构进行了表征，以确定主链运动对该结构域的DNA结合和脱氧核糖5'-磷酸裂解酶功能的潜在贡献。N端结构域由四个螺旋组成，以两个反平行对的形式堆积，两对之间有60度的交叉。NMR构象体（残基13 - 80）主链重原子的RMSD为0.37 Å，所有重原子的RMSD为0.78 Å。对单链DNA - 5聚体、单链DNA - 8聚体、单链DNA - 9聚体和双链DNA - 12聚体结合位点的NMR表征显示，这些不同的DNA寡聚体结合有一个共同表面，该表面围绕并包括脱氧核糖5'-磷酸裂解酶活性位点区域。螺旋1和2之间以及螺旋3和4之间的连接片段各自对DNA结合有贡献。螺旋3 - 转角 - 螺旋4形成一个螺旋 - 发夹 - 螺旋基序。高度保守的发夹序列（LPGVG）在主链内显示出显著程度的皮秒时间尺度运动，这可能对在磷酸二酯主链处的DNA结合很重要。连接螺旋1和2的Ω环以及螺旋2本身由于在毫秒时间尺度上明显的构象类型运动，在主链酰胺处显示出显著的交换贡献（R(ex)）。这种运动可能在允许Ω环和螺旋2向缺口DNA移动并与其有效相互作用方面很重要。包括形成席夫碱的K72、推测促进质子转移至醛的Y39以及协助磷酸消除的K35在内的脱氧核糖5'-磷酸裂解酶催化残基，显示出高度受限的主链运动。明显参与无碱基位点孔检测并协助半缩醛打开的H34，显示出构象交换。
