Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder |
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Authors: | van Oijen Antoine M Blainey Paul C Crampton Donald J Richardson Charles C Ellenberger Tom Xie X Sunney |
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Affiliation: | Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA. |
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Abstract: | We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda 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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