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.

SSB Binding to Single-Stranded DNA Probed Using Solid-State Nanopore Sensors

“…This is probably due to the translocation events being too fast and/or too small in magnitude to be detected. 36,39 In fact, the ratio between the volumes of SSB tet , V SSB = 5×5×8 nm 3 On the other hand, the translocation events could be detected for the complex and, under certain conditions, exhibited some interesting sub structure that warrants further analysis. Namely, for small biases (0.1 V), the translocation events could be subdivided into two classes, fig.…”

Glyphosate-induced stiffening of HaCaT keratinocytes, a Peak Force Tapping study on living cells

“…Indeed, AFM based force spectroscopy is increasingly used to study the mechanisms of molecular recognition and protein folding/unfolding, to probe chemical groups and dynamics of receptor-ligand interactions (Florin et al 1994;Dorobantu and Gray, 2010;Rico et al, 2011), and to study the local elasticity (Clausen-Schaumann et al, 2000) and the mechanical properties of soft biological samples (Butt et al, 2005;Müller and Dufrene, 2008). The AFM has also provided nanometer-scale resolution imaging of biological samples ranging from single molecules, such as DNA (Hamon et al, 2007), to intact cells attached on biomaterials (Berquand et al, 2010). Emerging applications include increasing the resolution of mechanical measurements in biological contexts such as cell division (Gilbert et al, 2007;Stewart et al, 2011) or cell adhesion (Li et al, 2003;Fierro et al, 2008).…”

Atomic Force Microscopy of Chromatin

“…Two DNA strands are usually entwined in a double helix stabilized by hydrogen bonds between bases on the different strands. DNA can present different conformation: relaxed or coiled, or complex forms during cellular process such as replication or recombination, which may be visualized on AFM (Barattin & Voyer, 2011;Hamon et al, 2007;Hansma & Laney, 1996;Hansma et al, 1995;Lyubchenko et al, 1992;Shlyakhtenko et al, 2003).…”