Dda Helicase Tightly Couples Translocation on Single-Stranded DNA to Unwinding of Duplex DNA: Dda Is an Optimally Active Helicase

JOURNAL OF MOLECULAR BIOLOGY

卷:420

Article

Byrd, Alicia K.²  Matlock, Dennis L.^1,3  Bagchi, Debjani⁴  Aarattuthodiyil, Suja²  Harrison, David²  Croquette, Vincent^1,4  Raney, Kevin D.² 

[1] Univ Paris 11 Diderot, Lab Phys Stat, CNRS, F-75005 Paris, France

[2] Univ Arkansas Med Sci, Dept Biochem & Mol Biol, Little Rock, AR 72205 USA

[3] Harding Univ, Dept Chem, Searcy, AR 72143 USA

[4] Univ Paris 11 Diderot, UPMC Univ Paris 06, CNRS, Ecole Normale Super,IBENS, F-75005 Paris, France

关键词: molecular motor;    single-molecule;    stopped-flow;    DNA;    fluorescence;   

DOI  :  10.1016/j.jmb.2012.04.007

来源: Elsevier

【 摘 要 】

Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V-un) by the velocity of translocation on ssDNA in nucleotides per second (V-trans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the active range. In the case of Dda, the average DNA unwinding velocity was 257 +/- 42 bp/s, and the average translocation velocity was 267 +/- 15 nt/s. The V-un/V-trans value of 0.96 places Dda in a unique category of being an essentially perfectly active helicase. (C) 2012 Elsevier Ltd. All rights reserved.

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