Kinetic study of various binding modes between human DNA polymerase β and different DNA substrates by surface-plasmon-resonance biosensor

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review

26 Scopus Citations
View graph of relations

Author(s)

Detail(s)

Original languageEnglish
Pages (from-to)317-325
Journal / PublicationBiochemical Journal
Volume361
Issue number2
Publication statusPublished - 15 Jan 2002

Abstract

The interaction of a series of DNA substrates with human DNA polymerase β 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 β 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 β binding to single-stranded DNA (KA = 1.25 × 108 M-1 ; where KA is the equilibrium affinity constant) was thermodynamically more favourable than to blunt-end DNA duplex (KA = 7.56 × 107 M-1) or gapped DNA (KA = 8.53 × 107 M-1), with a single binding mode on each DNA substrate. However, polymerase β 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 β (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 β bound at the duplex region to decrease 8-58-fold and 15-40%, respectively. On the other hand, the affinity of polymerase β 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 β.

Research Area(s)

  • DNA primer-template duplex, Human DNA polymerase β, Molecular recognition, Parallel binding, SPR biosensor