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

Biochemistry
S S MandalLinda Reha-Krantz

Abstract

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 sequ...Continue Reading

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Citations

Mar 1, 2006·Biochemistry·Chithra HariharanLinda J Reha-Krantz
Oct 6, 2010·Proceedings of the National Academy of Sciences of the United States of America·Kausiki DattaPeter H von Hippel
Sep 8, 2012·The Journal of Biological Chemistry·Kamalendra SinghStefan G Sarafianos
Aug 19, 2007·Nucleic Acids Research·Elizabeth Fidalgo da Silva, Linda J Reha-Krantz
Oct 16, 2010·Nucleic Acids Research·Sarah K Jarchow-ChoyEric T Kool
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Mar 3, 2004·Proceedings of the National Academy of Sciences of the United States of America·Neil P JohnsonPeter H Von Hippel
Dec 18, 2002·Proceedings of the National Academy of Sciences of the United States of America·Melanie A O'Neill, Jacqueline K Barton
May 11, 2005·Proceedings of the National Academy of Sciences of the United States of America·Neil P JohnsonPeter H von Hippel
Sep 13, 2006·Journal of Biomedical Optics·Evaldas Katilius, Neal W Woodbury
Jul 26, 2015·Biochimica Et Biophysica Acta·Arunoday Bhan, Subhrangsu S Mandal
Aug 20, 2014·Frontiers in Microbiology·Linda J Reha-KrantzMyron F Goodman
Oct 4, 2005·Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics·E HernándezA Mondragón
Aug 22, 2002·The Journal of Biological Chemistry·Elizabeth Fidalgo da SilvaLinda J Reha-Krantz
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May 2, 2007·The Journal of Physical Chemistry. B·T RamreddyG Krishnamoorthy
Oct 31, 2002·Journal of the American Chemical Society·Melanie A O'Neill, Jacqueline K Barton

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