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

被引:282
作者
van Oijen, AM
Blainey, PC
Crampton, DJ
Richardson, CC
Ellenberger, T
Xie, XS
机构
[1] Harvard Univ, Dept Chem & Biol Chem, Cambridge, MA 02138 USA
[2] Harvard Univ, Sch Med, Dept Biol Chem & Pharmacol, Boston, MA 02115 USA
关键词
D O I
10.1126/science.1084387
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:1235 / 1238
页数:4
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