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

Content Provider	Semantic Scholar
Author	Heijden, Thijn Van Der Moreno-Herrero, Fernando Kanaar, Roland Dekker, Cees
Copyright Year	2006
Abstract	Introduction. Recently, Li et al. published a paper entitled “Direct and real-time visualization of the disassembly of a single RecA-DNA-ATPγS complex using AFM imaging in fluid”. 1 They reported the disassembly of RecA from doublestranded (ds) DNA as observed by AFM imaging in deionized water. RecA plays an important role during homologous recombination forming a nucleoprotein filament that drives DNA strand invasion and exchange reactions. Li et al. drew two main conclusions from their work. First, RecA dissociates in a biologically relevant manner with greatly varying rates, and second, it dissociates as hexamers. We have performed similar experiments previously (presented below) and come to quite different conclusions. We imaged RecA filaments in buffered solutions where they are known to be biochemically active, as opposed to the deionized water used in the experiments of Li et al., where the biochemical activity of RecA is undefined. Our results, and we argue also the images presented by Li et al., indicate that RecA dissociation is strongly influenced by the scanning tip. The relevance of the work by Li et al. to the intrinsic biochemical activity of RecA is thus questionable. Furthermore, we address some concerns about image resolution and quantification that need to be considered in order to evaluate data on the size of objects in AFM images. Tip-Sample Interaction Strongly Influences RecA Filament Disassembly.The biochemical conditions described by Li et al. influence the RecA-DNA disassembly reaction in ways that were not discussed. They initiate the disassembly of RecA-dsDNA-ATP γS complexes by exchanging the bulk fluid in the experimental setup with deionized water. The biochemical activities of RecA in deionized water are, however, ill defined. Furthermore, the pH of deionized water is not stable because it is not buffered. Diffusion of CO2 into the fluid will drop the pH to about 5.5. The disassembly behavior of RecA from dsDNA is coupled to ATP hydrolysis and is highly pH-dependent. At a pH below 6.75, no net disassembly of RecA filaments from dsDNA is observed. 2 Because RecA is extremely slow in hydrolyzing ATPγS, disassembly is very inefficient, 3 as has been well established in single-molecule experiments. 4
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Alternate Webpage(s)	http://www.fernandomorenoherrero.com/pdf/Heijden_Nanoletters_2006.pdf
PubMed reference number	17163748v1
Volume Number	6
Issue Number	12
Journal	Nano letters
Language	English
Access Restriction	Open
Subject Keyword	Adenosine Triphosphate Auriculo-Ventricular Dissociation Buffers Carbon Dioxide Cytoskeletal Filaments DNA Repair Protein RAD51 Homolog 1 Disassembly (action) Ephrin Type-B Receptor 1, human Homologous Recombination Imagery Intrinsic drive Lithium Nucleoproteins Physical object Preparation Quantitation RAD51 gene RAD51 wt Allele Scientific Publication strand invasion
Content Type	Text
Resource Type	Article