Single-Molecule Kinetics of λ Exonuclease Reveal Base Dependence and Dynamic Disorder

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of λ-phage DNA by individual bacteriophage λ exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the l...

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Veröffentlicht in:Science (American Association for the Advancement of Science) 2003-08, Vol.301 (5637), p.1235-1238
Hauptverfasser: van Oijen, Antoine M., Blainey, Paul C., Crampton, Donald J., Richardson, Charles C., Ellenberger, Tom, Xie, X. Sunney
Format: Artikel
Sprache:eng
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Zusammenfassung:We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of λ-phage DNA by individual bacteriophage λ exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.1084387