Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase.
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.
Mandal
Subhrangsu S
SS
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Fidalgo da Silva
Elizabeth
E
Reha-Krantz
Linda J
LJ
D015075:2-Aminopurine / Q000494:pharmacology*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000963:Antimetabolites / Q000494:pharmacology*; D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005821:Genetic Techniques; D006157:Guanosine Monophosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry*
D000963:Antimetabolites; D005456:Fluorescent Dyes; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D000249:Adenosine Monophosphate; D015075:2-Aminopurine; D006157:Guanosine Monophosphate; D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase