Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I.
Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.
Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands. seidel@mb.tn.tudelft.nl
Ralf
R
Seidel
John
J
van Noort
Carsten
C
van der Scheer
Joost G P
JG
Bloom
Nynke H
NH
Dekker
Christina F
CF
Dutta
Alex
A
Blundell
Terence
T
Robinson
Keith
K
Firman
Cees
C
Dekker
D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D001692:Biological Transport; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D013997:Time Factors
D002843:Chromatin; D000255:Adenosine Triphosphate; D004247:DNA; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific