Analysis of histone chaperone antisilencing function 1 interactions.
The assembly and disassembly of chromatin impacts all DNA-dependent processes in eukaryotes. These processes are intricately regulated through stepwise mechanisms, requiring multiple proteins, posttranslational modifications, and remodeling enzymes, as well as specific proteins to chaperone the highly basic and aggregation-prone histone proteins. The histone chaperones are acidic proteins that perform the latter function by maintaining the stability of the histones when they are not associated with DNA and guiding the deposition and removal of histones from DNA. Understanding the thermodynamics of these processes provides deeper insights into the mechanisms of chromatin assembly and disassembly. Here we describe complementary thermodynamic and biochemical approaches for analysis of the interactions of a major chaperone of the H3/H4 dimer, anti-silencing function 1 (Asf1) with histones H3/H4, and DNA. Fluorescence quenching approaches are useful for measuring the binding affinity of Asf1 for histones H3/H4 under equilibrium conditions. Electrophoretic mobility shift analyses are useful for examining Asf1-mediated tetrasome (H3/H4-DNA) assembly and disassembly processes. These approaches potentially can be used more generally for the study of other histone chaperone-histone interactions and provide a means to dissect the role of posttranslational modifications and other factors that participate in chromatin dynamics.
Department of Pharmacology, University of Colorado, School of Medicine, Aurora, Colorado, USA.
Jean K
JK
Scorgie
Douglas C
DC
Donham
Mair E A
ME
Churchill
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D006657:Histones / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013816:Thermodynamics; D029867:Xenopus Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
C109731:ASF1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D017931:DNA Primers; D006657:Histones; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins