Direct visualization of dynamic protein-DNA interactions with a dedicated atomic force microscope.
Photolyase DNA interactions and the annealing of restriction fragment ends are directly visualized with the atomic force microscope (AFM). To be able to interact with proteins, DNA must be loosely bound to the surface. When MgCl2 is used to immobilize DNA to mica, DNA is attached to the surface at distinct sites. The pieces of DNA in between are free to move over the surface and are available for protein interaction. After implementation of a number of instrumental improvements, the molecules can be visualized routinely, under physiological conditions and with molecular resolution. Images are acquired reproducibly without visible damage for at least 30 min, at a scan rate of 2 x 2 microm2/min and a root mean square noise of less than 0.2 nm. Nonspecific photolyase DNA complexes were visualized, showing association, dissociation, and movement of photolyase over the DNA. The latter result suggests a sliding mechanism by which photolyase can scan DNA for damaged sites. The experiments illustrate the potential that AFM presents for modern molecular biology.
van Noort
S J
SJ
Department of Applied Physics, University of Twente, Enschede, The Netherlands. s.j.t.vannoort@tn.utwente.nl
van der Werf
K O
KO
Eker
A P
AP
Wyman
C
C
de Grooth
B G
BG
van Hulst
N F
NF
Greve
J
J
D001665:Binding Sites; D000458:Cyanobacteria / Q000201:enzymology; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D004867:Equipment Design; D018625:Microscopy, Atomic Force / Q000295:instrumentation* / Q000379:methods; D009068:Movement; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D012680:Sensitivity and Specificity
D011506:Proteins; D004247:DNA; D004255:Deoxyribodipyrimidine Photo-Lyase