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Using 2-Aminopurine Fluorescence To Detect Base Unstacking in the Template Strand during Nucleotide Incorporation by the Bacteriophage T4 DNA Polymerase

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Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
Cite this: Biochemistry 2002, 41, 13, 4399–4406
Publication Date (Web):March 5, 2002
Copyright © 2002 American Chemical Society

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    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.

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     This work was supported by Canadian Institutes of Health Research Grant 14300 (to L.J.R.-K.), by an Alberta Heritage Foundation for Medical Research postdoctoral fellowship (to S.S.M.), and by CAPES (Brasilia) and the Universidade Gama Filho, Rio de Janeiro, Brazil (to E.F.S.). L.J.R.-K. is a Scientist of the Alberta Heritage Foundation for Medical Research.

     These authors contributed equally to the work.


     To whom correspondence should be addressed:  CW405 Biological Sciences Building, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. Phone:  (780) 492-5383. Fax:  (780) 492-2216. E-mail:  [email protected].

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