Skip to main content
placeholder image

Single-molecule kinetics of ¿ exonuclease reveal base dependence and dynamic disorder

Journal Article


Abstract


  • 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 enzym e-DNA complex.

Publication Date


  • 2003

Citation


  • Van Oijen, A. M., Blainey, P. C., Crampton, D. J., Richardson, C. C., Ellenberger, T., & Xie, X. S. (2003). Single-molecule kinetics of ¿ exonuclease reveal base dependence and dynamic disorder. Science, 301(5637), 1235-1238. doi:10.1126/science.1084387

Scopus Eid


  • 2-s2.0-0041821716

Start Page


  • 1235

End Page


  • 1238

Volume


  • 301

Issue


  • 5637

Abstract


  • 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 enzym e-DNA complex.

Publication Date


  • 2003

Citation


  • Van Oijen, A. M., Blainey, P. C., Crampton, D. J., Richardson, C. C., Ellenberger, T., & Xie, X. S. (2003). Single-molecule kinetics of ¿ exonuclease reveal base dependence and dynamic disorder. Science, 301(5637), 1235-1238. doi:10.1126/science.1084387

Scopus Eid


  • 2-s2.0-0041821716

Start Page


  • 1235

End Page


  • 1238

Volume


  • 301

Issue


  • 5637