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 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 = 7.56 x 10(1) M-1) or gapped DNA (KA = 8.53 x 10(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 m 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 the binding affinity and the fraction of polymerase beta bound at the duplex region. The introduction of different numbers of mismatches near the 3'-end of the primer caused duplex region to decrease 8-58-fold and 15-40%, respethe binding affinity and the fraction of polymerase beta bound at the 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.