Stopped-flow fluorescence study of precatalytic primer strand base-unstacking transitions in the exonuclease cleft of bacteriophage T4 DNA polymerase.
DNA polymerases are complex enzymes which bind primer-template DNA and subsequently either extend or excise the terminal nucleotide on the primer strand. In this study, a stopped-flow fluorescence anisotropy binding assay is combined with real-time measurements of a fluorescent adenine analogue (2-aminopurine) located at the 3'-primer terminus. Using this combined approach, the exact time course associated with protein binding, primer terminus unstacking, and base excision by the 3' --> 5' exonuclease of bacteriophage T4 (T4 pol) was examined. T4 pol binding and dissociation kinetics were found to obey simple kinetics, with identical on rates (kon = 4.6 x 10(8) M-1 s-1) and off rates (koff = 9.3 s-1) for both single-stranded primers and double-stranded primer-templates (at 100 microM Mg2+). Although the time course for T4 pol-DNA association and dissociation obeyed simple kinetics, at suboptimal Mg2+ concentrations (e.g., 100 microM), non-first-order sigmoidal kinetics were observed for the base-unstacking reaction of the primer terminus in double-stranded primer-templates. The observed sigmoidal kinetics for base unstacking demonstrate that T4 pol is a hysteretic enzyme [Frieden, C. (1970) J. Biol. Chem. 245, 5788-5799] and must exist in two DNA bound conformations which differ greatly in base-unstacking properties. A Mg2+-dependent time lag of 10 ms is observed between primer-template binding and the beginning of the unstacking transition, which is 50% complete at 22 +/- 1 ms after addition of 100 microM Mg2+. Following the hysteretic lag, a simple first-order primer terminus unstacking rate of 130 s-1 is resolved, which is protein and Mg2+ concentration-independent. For the processing of single-stranded primers, all kinetic complexity is lost, and T4 pol binding and primer end base-unstacking kinetics can be superimposed. These data reveal that the kinetic processing of double-stranded primer-template DNA by T4 pol is much more complex than that of single-stranded primers, and suggest that the intrinsic "switching rate" between the polymerase and exonuclease sites may be much faster than previously proposed.
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
M R
MR
Otto
L B
LB
Bloom
M F
MF
Goodman
J M
JM
Beechem
D015075:2-Aminopurine / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D015075:2-Aminopurine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D008274:Magnesium