Surface plasmon resonance study of DNA polymerases binding to template/primer DNA duplexes immobilized on supported lipid monolayers

Pui Yan Tsoi, Jun Yang, Yutong Sun, Sen Fang Sui, Mengsu Yang

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Recent advances in biosensor technology have contributed significantly to our understanding of the mechanisms of molecular recognition processes. In this study, a surface plasmon resonance (SPR)-based optical sensor was employed to study the binding interactions between a series of template/primer double-stranded DNA (dsDNA) and DNA polymerase enzymes containing both polymerase and exonuclease activities. The DNA molecules were immobilized on the SPR gold surface by the controlled assembly of a biotinylated phospholipid monolayer, an avidin monolayer, and a layer of biotinylated DNA. By introducing a number of different mismatches at/near the 3′-end of the primer, the effects of the mismatches on the overall binding affinities of the Klenow fragment (KF) of Escherichia coli DNA polymerase I and T4 and T7 bacteriophage polymerases were measured. While no obvious trend in the overall binding affinity of the Klenow fragment was observed with the introduction of mismatched base pair(s), its polymerase domain binds with the fully matched DNA 20 times more strongly than with the DNA containing three consecutive terminal mismatches. On the other hand, its exonuclease domain binds with the DNA containing three terminal mismatches 12 times more strongly than with the fully matched DNA. For both T4 and T7 polymerases, the overall affinities of the enzymes toward DNA increased as the number of mismatches increased, consistent with previous reports that the enhanced melting ability of terminally mismatched DNA duplex leads to the preferential binding at the exonuclease site. The results have demonstrated the feasibility of using the SPR biosensor to study molecular recognition events such as single base discrimination involved in protein-DNA interactions.

Original languageEnglish (US)
Pages (from-to)6590-6596
Number of pages7
JournalLangmuir
Volume16
Issue number16
DOIs
StatePublished - Aug 8 2000

Fingerprint

primers
Exonucleases
Surface plasmon resonance
surface plasmon resonance
Lipids
lipids
Monolayers
Molecular recognition
DNA Polymerase I
templates
affinity
Biosensors
Enzymes
bioinstrumentation
enzymes
Bacteriophages
Avidin
Phospholipids
Optical sensors
fragments

ASJC Scopus subject areas

  • Materials Science(all)
  • Condensed Matter Physics
  • Surfaces and Interfaces
  • Spectroscopy
  • Electrochemistry

Cite this

Surface plasmon resonance study of DNA polymerases binding to template/primer DNA duplexes immobilized on supported lipid monolayers. / Tsoi, Pui Yan; Yang, Jun; Sun, Yutong; Sui, Sen Fang; Yang, Mengsu.

In: Langmuir, Vol. 16, No. 16, 08.08.2000, p. 6590-6596.

Research output: Contribution to journalArticle

Tsoi, Pui Yan ; Yang, Jun ; Sun, Yutong ; Sui, Sen Fang ; Yang, Mengsu. / Surface plasmon resonance study of DNA polymerases binding to template/primer DNA duplexes immobilized on supported lipid monolayers. In: Langmuir. 2000 ; Vol. 16, No. 16. pp. 6590-6596.
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