Alternative geometries of DNA looping: an analysis using the SfiI endonuclease

Many processes are governed by proteins that bind to separate sites in DNA and loop out the intervening DNA, but the geometries of the loops have seld om been determined. The SfiI endonuclease cleaves DNA after interacting wit h two recognition sites, and is a favourable system for the analysis of DNA looping. A gel-shift assay was used here to examine the binding of SfiI to a series of Linear DNA molecules containing two SfiI sites separated by 10 9-170 base-pairs. The complexes in which SfiI trapped a loop by binding to two sites in the same DNA were separated from the complexes containing SfiI bound to separate DNA molecules. Step-wise changes in the inter-site spaci ng generated two forms of the looped complex with different electrophoretic mobilities. The yields of each looped complex and the complexes from inter molecular synapses all varied cyclically with the inter-site spacing, with similar periodicities (similar to 10.5 base-pairs) but with different phase s. One looped complex predominated whenever the DNA between the sites neede d to be underwound in order to produce the correct helical orientation of t he binding sites. The other looped complex predominated whenever the interv ening DNA needed to be overwound. We conclude that the former has trapped a right-handed loop with a negative node and the latter a left-handed loop w ith a positive node. (C) 2000 Academic Press.