Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase

Biochemistry. 2002 Apr 2;41(13):4399-406. doi: 10.1021/bi015723p.


The fluorescence of the base analogue 2-aminopurine (2AP) was used to detect physical changes in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Fluorescent enzyme-DNA complexes were formed with 2AP placed in the template strand opposite the primer terminus (the n position) and placed one template position 5' to the primer terminus (the n + 1 position). The fluorescence enhancement for 2AP at the n position was shown to be due to formation of the editing complex, which indicates that the 2AP-T terminal base pair is recognized primarily as a mismatch. 2AP fluorescence at the n + 1 position, however, was a reporter for DNA interactions in the polymerase active center that induce intrastrand base unstacking. T4 DNA polymerase produced base unstacking at the n + 1 position following formation of the phosphodiester bond. Thus, the increase in fluorescence intensity for 2AP at the n + 1 position could be used to measure the nucleotide incorporation rate in primer extension reactions in which 2AP was placed initially at the n + 2 position. Primer extension occurred at the rate of about 314 s(-1). The amount of base unstacking at the template n + 1 position was sensitive to the local DNA sequence. More base unstacking was detected for DNA substrates with an A-T base pair at the primer terminus compared to C-G or G-C base pairs. Since proofreading is also increased by A-T base pairs compared to G-C base pairs at the primer terminus, we propose that base unstacking may provide an opportunity for the DNA polymerase to reexamine the primer terminus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Aminopurine / pharmacology*
  • Adenosine Monophosphate / metabolism
  • Adenosine Triphosphate / pharmacology
  • Antimetabolites / pharmacology*
  • Base Pairing*
  • Base Sequence
  • DNA / metabolism
  • DNA-Directed DNA Polymerase*
  • Dose-Response Relationship, Drug
  • Fluorescent Dyes / pharmacology*
  • Genetic Techniques
  • Guanosine Monophosphate / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Models, Chemical
  • Molecular Sequence Data
  • Protein Binding
  • Spectrometry, Fluorescence
  • Spectrophotometry
  • Time Factors
  • Viral Proteins / chemistry*


  • Antimetabolites
  • Fluorescent Dyes
  • Viral Proteins
  • gene 43 protein, Enterobacteria phage T4
  • Adenosine Monophosphate
  • 2-Aminopurine
  • Guanosine Monophosphate
  • Adenosine Triphosphate
  • DNA
  • DNA-Directed DNA Polymerase