Journal
SCIENCE
Volume 301, Issue 5637, Pages 1235-1238Publisher
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/science.1084387
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- NIGMS NIH HHS [5R01GM61577-03, R01GM55390-07] Funding Source: Medline
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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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