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

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 (K_A = 1.25 × 10⁸ M^-1 ; where K_A is the equilibrium affinity constant) was thermodynamically more favourable than to blunt-end DNA duplex (K_A = 7.56 × 10⁷ M^-1) or gapped DNA (K_A = 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.