Escherichia coli RecQ is a rapid, efficient, and monomeric helicase.
RecQ family helicases play a key role in chromosome maintenance. Despite extensive biochemical, biophysical, and structural studies, the mechanism by which helicase unwinds double-stranded DNA remains to be elucidated. Using a wide array of biochemical and biophysical approaches, we have previously shown that the Escherichia coli RecQ helicase functions as a monomer. In this study, we have further characterized the kinetic mechanism of the RecQ-catalyzed unwinding of duplex DNA using the fluorometric stopped-flow method based on fluorescence resonance energy transfer. Our results show that RecQ helicase binds preferentially to 3'-flanking duplex DNA. Under the pre-steady-state conditions, the burst amplitude reveals a 1:1 ratio between RecQ and DNA substrate, suggesting that an active monomeric form of RecQ helicase is involved in the catalysis. Under the single-turnover conditions, the RecQ-catalyzed unwinding is independent of the 3'-tail length, indicating that functional interactions between RecQ molecules are not implicated in the DNA unwinding. It was further determined that RecQ unwinds DNA rapidly with a step size of 4 bp and a rate of approximately 21 steps/s. These kinetic results not only further support our previous conclusion that E. coli RecQ functions as a monomer but also suggest that some of the Superfamily 2 helicases may function through an "inchworm" mechanism.
Zhang
Xing-Dong
XD
Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China.
Dou
Shuo-Xing
SX
Xie
Ping
P
Hu
Jin-Shan
JS
Wang
Peng-Ye
PY
Xi
Xu Guang
XG
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D016880:Anisotropy; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D053484:RecQ Helicases
D004277:DNA, Single-Stranded; D004247:DNA; D000251:Adenosine Triphosphatases; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases