title: Kinetic study of various binding modes between human DNA polymerase beta and different DNA substrates by surface-plasmon-resonance biosensor.
abstract: 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.
authors.affiliation: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, People's Republic of China.
authors.forename: Pui Yan
authors.initials: PY
authors.lastname: Tsoi
authors.forename: Mengsu
authors.initials: M
authors.lastname: Yang
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D019951:DNA Polymerase beta / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000295:instrumentation*; D013698:Templates, Genetic
chemical_list: D017931:DNA Primers; D019951:DNA Polymerase beta