Initiation of translocation by Type I restriction-modification enzymes is associated with a short DNA extrusion.
Recognition of 'foreign' DNA by Type I restriction-modification (R-M) enzymes elicits an ATP-dependent switch from methylase to endonuclease activity, which involves DNA translocation by the restriction subunit HsdR. Type I R-M enzymes are composed of three (Hsd) subunits with a stoichiometry of HsdR2:HsdM2:HsdS1 (R2-complex). However, the EcoR124I R-M enzyme can also exist as a cleavage deficient, sub-assembly of HsdR1:HsdM2:HsdS1 (R1-complex). ATPS was used to trap initial translocation complexes, which were visualized by Atomic Force Microscopy (AFM). In the R1-complex, a small bulge, associated with a shortening in the contour-length of the DNA of 8 nm, was observed. This bulge was found to be sensitive to single-strand DNA nucleases, indicative of non-duplexed DNA. R2-complexes appeared larger in the AFM images and the DNA contour length showed a shortening of approximately 11 nm, suggesting that two bulges were formed. Disclosure of the structure of the first stage after the recognition-translocation switch of Type I restriction enzymes forms an important first step in resolving a detailed mechanistic picture of DNA translocation by SF-II DNA translocation motors.
van Noort
John
J
Delft University of Technology, Faculty of Applied Sciences and Dimes, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
van der Heijden
Thijn
T
Dutta
Christina F
CF
Firman
Keith
K
Dekker
Cees
C
D001692:Biological Transport; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation
D004277:DNA, Single-Stranded; D004247:DNA; D004706:Endodeoxyribonucleases; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific