Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I.
Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino-acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single-molecule magnetic tweezers and stopped-flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation steps--ATP binding and extrusion of an initial DNA loop--allowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.
Seidel
Ralf
R
Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.
Bloom
Joost G P
JG
van Noort
John
J
Dutta
Christina F
CF
Dekker
Nynke H
NH
Firman
Keith
K
Szczelkun
Mark D
MD
Dekker
Cees
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active / Q000502:physiology; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D007700:Kinetics; D021381:Protein Transport
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D004248:DNA (Cytosine-5-)-Methyltransferases; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific; C109830:HsdR protein, E coli