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It has been known for some time that addition of a non-hydrolysable analogue of ATP (e.g. ATPgS) not only prevents translocation of the DNA, but also 'freezes' the initiation complex. Therefore,
van Noort et al (2004) used ATPgS
to trap initial translocation complexes, which were visualized
by Atomic Force Microscopy (AFM). In the R1-complex,
a small bulge was observed using atomic force microscopy (AFM),
that is associated with a shortening in the contour-length of
the DNA by 8 nm. This bulge was found to be sensitive to
single-strand |
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• © University of
Portsmouth |
DNA nucleases, indicative of non-duplexed DNA. R2-complexes
appeared larger in the AFM images and the DNA contour length
showed a shortening of 11 nm, suggesting that two bulges were
formed; although the images were unable to resolve such a
structure. Disclosure of this structure at 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 (