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

作者：Antoine M. van Oijen, Paul C. Blainey, Donald J. Crampton, Charles Clifton Richardson, Tom Ellenberger, Xiaohui S. Xie · 发表于：Science · 年份：2003 · DOI：10.1126/science.1084387 · 被引用次数：378 · 研究领域：DNA and Nucleic Acid Chemistry、Nanopore and Nanochannel Transport Studies、Advanced biosensing and bioanalysis techniques

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.