title: Protein-DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54.
abstract: High-speed AFM (atomic force microscopy also called scanning force microscopy) provides nanometre spatial resolution and sub-second temporal resolution images of individual molecules. We exploit these features to study diffusion and motor activity of the RAD54 DNA repair factor. Human RAD54 functions at critical steps in recombinational-DNA repair. It is a member of the Swi2/Snf2 family of chromatin remodelers that translocate on DNA using ATP hydrolysis. A detailed single molecular description of DNA-protein interactions shows intermediate states and distribution of variable states, usually hidden by ensemble averaging. We measured the motion of individual proteins using single-particle tracking and observed that random walks were affected by imaging-buffer composition. Non-Brownian diffusion events were characterized in the presence and in the absence of nucleotide cofactors. Double-stranded DNA immobilized on the surface functioned as a trap reducing Brownian motion. Distinct short range slides and hops on DNA were visualized by high-speed AFM. These short-range interactions were usually inaccessible by other methods based on optical resolution. RAD54 monomers displayed a diffusive behavior unrelated to the motor activity.
authors: Department of Cell Biology and Genetics, Cancer Genomics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands. h.sanchezgonzalez@erasmusmc.nl
authors: Humberto
authors: H
authors: Sanchez
authors: Yuki
authors: Y
authors: Suzuki
authors: Masatoshi
authors: M
authors: Yokokawa
authors: Kunio
authors: K
authors: Takeyasu
authors: Claire
authors: C
authors: Wyman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000465:Algorithms; D000538:Aluminum Silicates / Q000378:metabolism; D002021:Buffers; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004058:Diffusion / Q000187:drug effects*; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D018625:Microscopy, Atomic Force / Q000379:methods*; D009038:Motion; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000538:Aluminum Silicates; D002021:Buffers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; C103532:RAD54L protein, human; C011934:mica