DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies.
The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.
Lucius
Aaron L
AL
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., Box 8231, St. Louis, MO 63110, USA.
Vindigni
Alessandro
A
Gregorian
Razmic
R
Ali
Janid A
JA
Taylor
Andrew F
AF
Smith
Gerald R
GR
Lohman
Timothy M
TM
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases