Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping.
Atomic force microscopy (AFM) allows the study of single protein-DNA interactions such as those observed with the Type I Restriction-Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPgammaS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein-DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.
Neaves
Kelly J
KJ
Department of Pharmacology, University of Cambridge, Cambridge, UK.
Cooper
Laurie P
LP
White
John H
JH
Carnally
Stewart M
SM
Dryden
David T F
DT
Edwardson
J Michael
JM
Henderson
Robert M
RM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D004262:DNA Restriction Enzymes / Q000378:metabolism / Q000648:ultrastructure*; D003627:Data Interpretation, Statistical; D019281:Dimerization; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D055503:Protein Multimerization; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism / Q000648:ultrastructure
C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; C093244:DNA modification methylase EcoKI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes; C089629:endodeoxyribonuclease EcoKI