title: High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein--DNA complexes.
abstract: DNA in living cells is generally processed via the generation and the protection of single-stranded DNA involving the binding of ssDNA-binding proteins (SSBs). The studies of SSB-binding mode transition and cooperativity are therefore critical to many cellular processes like DNA repair and replication. However, only a few atomic force microscopy (AFM) investigations of ssDNA nucleoprotein filaments have been conducted so far. The point is that adsorption of ssDN A-SSB complexes on mica, necessary for AFM imaging, is not an easy task. Here, we addressed this issue by using spermidine as a binding agent. This trivalent cation induces a stronger adsorption on mica than divalent cations, which are commonly used by AFM users but are ineffective in the adsorption of ssDNA-SSB complexes. At low spermidine concentration (<0.3 mM), we obtained AFM images of ssDNA-SSB complexes (E. coli SSB, gp32 and yRPA) on mica at both low and high ionic strengths. In addition, partially or fully saturated nucleoprotein filaments were studied at various monovalent salt concentrations thus allowing the observation of SSB-binding mode transition. In association with conventional biochemical techniques, this work should make it possible to study the dynamics of DNA processes involving DNA-SSB complexes as intermediates by AFM.
authors: Laboratoire de Structure et Activité des Biomolécules Normales et Pathologiques, INSERM U829, Université d'Evry-Val d'Essonne EA3637, Evry, F-91025, France. loic.hamon@univ-evry.fr
authors: Loïc
authors: L
authors: Hamon
authors: David
authors: D
authors: Pastré
authors: Pauline
authors: P
authors: Dupaigne
authors: Cyrille
authors: C
authors: Le Breton
authors: Eric
authors: E
authors: Le Cam
authors: Olivier
authors: O
authors: Piétrement
mesh_terms: D000538:Aluminum Silicates / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000648:ultrastructure*; D004587:Electrophoresis, Agar Gel; D029968:Escherichia coli Proteins / Q000648:ultrastructure; D018625:Microscopy, Atomic Force / Q000379:methods*; D013095:Spermidine / Q000737:chemistry
chemical_list: D000538:Aluminum Silicates; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D013095:Spermidine; C011934:mica