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

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Science  29 Aug 2003:
Vol. 301, Issue 5637, pp. 1235-1238
DOI: 10.1126/science.1084387

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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.

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