Tsoi Pui Yan, Yang Mengsu
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, People's Republic of China.
Biochem J. 2002 Jan 15;361(Pt 2):317-25. doi: 10.1042/0264-6021:3610317.
The interaction of a series of DNA substrates with human DNA polymerase beta has been studied in real time by using a surface-plasmon-resonance (SPR) biosensor technique. We have prepared the sensor surfaces comprising different DNA targets, including single-stranded DNA, blunt-end double-stranded DNA, gapped DNA and DNA template-primer duplexes containing various mismatches at different positions. The binding and dissociation of polymerase beta at the DNA-modified surfaces was measured in real time, and the kinetics profiles of polymerase-DNA interaction were analysed using various physical models. The results showed that polymerase beta binding to single-stranded DNA (K(A)=1.25 x 10(8) M(-1); where K(A) is the equilibrium affinity constant) was thermodynamically more favourable than to blunt-end DNA duplex (K(A)=7.56x10(7) M(-1)) or gapped DNA (K(A)=8.53x10(7) M(-1)), with a single binding mode on each DNA substrate. However, polymerase beta bound to DNA template-primer duplexes (15 bp with a 35 nt overhang) at two sites, presumably one at the single-strand overhang and the other at the 3'-end of the primer. When the DNA duplex was fully matched, most of the polymerase beta (83%) bound to the template-primer duplex region. The introduction of different numbers of mismatches near the 3'-end of the primer caused the binding affinity and the fraction of polymerase beta bound at the duplex region to decrease 8-58-fold and 15-40%, respectively. On the other hand, the affinity of polymerase beta for the single-strand overhang remained unchanged while the fraction bound to the single-strand region increased by 15-40%. The destabilizing effect of the mismatches was due to both a decrease in the rate of binding and an increase in the rate of dissociation for polymerase beta.
利用表面等离子体共振（SPR）生物传感器技术实时研究了一系列DNA底物与人类DNA聚合酶β的相互作用。我们制备了包含不同DNA靶标的传感器表面，包括单链DNA、平端双链DNA、缺口DNA以及在不同位置含有各种错配的DNA模板-引物双链体。实时测量了聚合酶β在DNA修饰表面的结合和解离，并使用各种物理模型分析了聚合酶-DNA相互作用的动力学曲线。结果表明，聚合酶β与单链DNA结合（K(A)=1.25×10(8) M(-1)；其中K(A)是平衡亲和常数）在热力学上比与平端DNA双链体（K(A)=7.56×10(7) M(-1)）或缺口DNA（K(A)=8.53×10(7) M(-1)）结合更有利，且在每种DNA底物上具有单一结合模式。然而，聚合酶β在两个位点与DNA模板-引物双链体（15 bp，带有35 nt突出端）结合，推测一个位点在单链突出端，另一个位点在引物的3'端。当DNA双链完全匹配时，大部分聚合酶β（83%）与模板-引物双链区域结合。在引物3'端附近引入不同数量的错配会导致结合亲和力以及在双链区域结合的聚合酶β比例分别降低8 - 58倍和15 - 40%。另一方面，聚合酶β对单链突出端的亲和力保持不变，而与单链区域结合的比例增加了15 - 40%。错配的去稳定作用是由于聚合酶β的结合速率降低和解离速率增加所致。
