Kinetic study of various binding modes between human DNA polymerase beta 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)=7.56x10(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.
Tsoi
Pui Yan
PY
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, People's Republic of China.
Yang
Mengsu
M
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
D017931:DNA Primers; D019951:DNA Polymerase beta