Biomedical subjects

Bernie D Sattin

Publications and source records attributed to Bernie D Sattin.

3 recordsLinked to original sources

PubMed2006

Direct and real-time visualization of the disassembly of a single RecA-DNA-ATPgammaS complex using AFM imaging in fluid.

RecA disassembly from circular double-stranded DNA (dsDNA) was studied by atomic force microscopy (AFM) imaging in fluid on a single molecule scale. The RecA/DNA complex was formed in the presence of ATPgammaS, and the disassembly was then initiated by buffer exchange to rinse off ATPgammaS. Performing AFM imaging in fluid allowed direct and real-time visualization of the disassembly of RecA from dsDNA in solution. It was found that RecA disassembly commenced from multiple sites both in deionized water and in buffer; the areas where RecA dissociated showed the appearance of "gaps" in the filamentous structure. RecA further disassembled either through the already existing "gaps" or by generation of new gaps. The disassembly was slower in buffer than in deionized water, suggesting that ions also contribute to the stabilization of the complex. RecA hexamers and monomers were observed in deionized water and in buffer, respectively, during the disassembly process.

Adenosine Triphosphate↗

PubMed2004

DNA base pair resolution by single molecule force spectroscopy.

The forces that hold complementary strands of DNA together in a double helix, and the role of base mismatches in these, are examined by single molecule force spectroscopy using an atomic force microscope (AFM). These forces are important when considering the binding of proteins to DNA, since these proteins often mechanically stretch the DNA during their action. In AFM measurement of forces, there is an inherent instrumental limitation that makes it difficult to compare results from different experimental runs. This is circumvented by using an oligonucleotide microarray, which allowed a direct comparison of the forces between perfectly matched short oligonucleotides and those containing a single or double mismatch. Through this greatly increased sensitivity, the force contribution of a single AT base pair was derived. The results indicate that the contribution to forces from the stacking interactions is more important than that from hydrogen bonding.

Base Pair Mismatch↗

PubMed2004

Direct observation of the assembly of RecA/DNA complexes by atomic force microscopy.

The formation of the RecA/DNA nucleofilament on nicked circular double stranded (ds) DNA in the presence of ATPgammaS was studied using the atomic force microscope (AFM) at nanometer resolution. The AFM allowed simultaneous observation of both dsDNA substrate and RecA protein-coated sections such that they are highly distinguishable. Using a time series of images, the complex formation was monitored. AFM imaging provided direct evidence that assembly of the nucleofilaments occurs via a nucleation and growth mechanism. The nucleation step is much slower than the growth phase, as demonstrated by the predominance of naked dsDNA at early and middle time points, followed by the rapid appearance of partially then fully formed complexes. Observation of the formation of nucleation sites without accompanying growth on unnicked dsDNA enabled an estimate of the nucleation rate, of 5 x 10(-5) RecA min(-1) bp(-1). The published model for the analysis of RecA assembly on dsDNA deduces a single kinetic parameter that prevents the separate determination of nucleation rate and growth rate. By directly measuring the nucleation rate with the AFM, this model is employed to determine a growth rate of 202 min(-1). These AFM results provide the first direct evidence of previous results on complex formation obtained only by indirect means.

Binding Sites↗