Direct observation of anomalous single-molecule enzyme kinetics.
We report the direct measurement of the single-molecule enzymatic cleavage rates of ApaI-DNA complex in the presence of various concentrations of MgCl2 solution with total internal reflection fluorescence microscopy. We made use of the native adsorption properties of the two 12-base sticky ends of the DNA molecules to partially immobilize and stretch out the ApaI-DNA complex onto a glass surface. Synchronous initiation of reaction was achieved by the influx of Mg2+ solution. Once the DNA was cut, the two fragments (38 and 10 kb) would either collapse or further stretch out depending on the solution flow. The time required for cleaving each ApaI-lambda-DNA complex was recorded and analyzed. At low concentrations, the higher the concentration of Mg2+, the faster the DNA was cut. However, Mg2+ ion is no longer the limiting factor when its concentration is greater than 5 mM. A surprising result is that at all concentrations the decrease in intact DNA population as a function of time is linear rather than exponential. This suggests that there exists a distribution of ApaI conformations around the restriction site.
Li
Hung-Wing
HW
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
Yeung
Edward S
ES
D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology
D015636:Magnesium Chloride; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C038485:GGGCCC-specific type II deoxyribonucleases