title: Allosteric interactions of DNA and nucleotides with S. cerevisiae RSC.
abstract: RSC (remodel the structure of chromatin) is an essential chromatin remodeler of Saccharomyces cerevisiae that has been shown to have DNA translocase properties. We studied the DNA binding properties of a "trimeric minimal RSC" (RSCt) of the RSC chromatin remodeling complex and the effect of nucleotides on this interaction using fluorescence anisotropy. RSCt binds to 20 bp fluorescein-labeled double-stranded DNA with a K(d) of ∼100 nM. The affinity of RSCt for DNA is reduced in the presence of AMP-PNP and ADP in a concentration-dependent manner with the addition of AMP-PNP having more pronounced effect. These differences in the magnitude at which the binding of ADP and AMP-PNP affects the affinity of DNA binding by RSCt suggest that the physical movement of the enzyme along DNA begins between the binding of ATP and its subsequent hydrolysis. Furthermore, the fact that the highest affinity for DNA binding by RSCt occurs in the absence of bound nucleotide offers a mechanistic explanation for the apparent low processivity of DNA translocation by the enzyme.
authors.affiliation: Department of Physics and Astronomy, University of Kansas, 1251 Wescoe Hall Dr., 1082 Malott Hall, Lawrence, Kansas 66045, USA.
authors.forename: Shuja Shafi
authors.initials: SS
authors.lastname: Malik
authors.forename: Evan
authors.initials: E
authors.lastname: Rich
authors.forename: Ramya
authors.initials: R
authors.lastname: Viswanathan
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Cairns
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fischer
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D042002:Chromatin Assembly and Disassembly / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009711:Nucleotides; C103337:RSC complex, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors