Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates.
We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5' to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3' hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.
Zhang
H
H
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, CT 06520, USA.
Cao
W
W
Zakharova
E
E
Konigsberg
W
W
De La Cruz
E M
EM
D015075:2-Aminopurine / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D017931:DNA Primers; D003854:Deoxyribonucleotides; D010710:Phosphates; D014764:Viral Proteins; D015075:2-Aminopurine; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase