Dna Binding and Translocation by S. Cerevisiae RSC

Abstract

We have studied the mechanisms of double-stranded DNA binding and double-stranded DNA translocation by a truncated construct of the RSC chromatin remodeling complex from S. cerevisiae. We monitored the double-stranded DNA translocation activity of RSC through its ability to actively displace streptavidin from biotin labeled double-stranded DNA. This displacement activity is ATP-dependent and is correlated with the translocation of the complex along the double-stranded DNA. We have also characterized the double-stranded DNA translocation of RSC through the use of a fluorescence-based stopped-flow assay in which the translocation of the complex is monitored through the interactions of the complex with fluorophores attached to the ends of the DNA. The kinetics of double-stranded DNA binding were also monitored using a fluorescence-based stopped-flow assay. The results indicate that double-stranded DNA binding occurs through a two-step mechanism, similar to what has been reported for other chromatin remodeling complexes and genetically related helicases.