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.
Otto
M R
MR
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
Bloom
L B
LB
Goodman
M F
MF
Beechem
J M
JM
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