PMID:16584187

Citation	Lee, AM and Singleton, SF (2006) Intersubunit electrostatic complementarity in the RecA nucleoprotein filament regulates nucleotide substrate specificity and conformational activation. Biochemistry 45:4514-29
Abstract	The Escherichia coli RecA protein is the prototypical member of a family of molecular motors that transduces ATP binding and hydrolysis for mechanical function. While many general mechanistic features of RecA action are known, specific structural and functional insights into the molecular basis of RecA activation remain elusive. Toward a more complete understanding of the interdependence between ATP and DNA binding by RecA, we report the characterization of a mutant RecA protein wherein the aspartate residue at position 100 within the ATP binding site is replaced by arginine. Physiologically, D100R RecA was characterized by an inducible, albeit reduced, activation of the SOS response and a diminished ability to promote cellular survival after UV irradiation. Biochemically, the D100R substitution caused a surprisingly modest perturbation of RecA-ATP interactions and an unexpected and significant decrease in the affinity of RecA for ssDNA. Moreover, in vitro assays of RecA activities requiring the coordinated processing of ATP and DNA revealed (1) a 2-5-fold decrease in steady-state turnover of ATP; (2) no formation of mixed nucleoprotein filaments when wild-type and D100R RecA compete for limiting ssDNA; and (3) no formation of strand exchange reaction products. Taken together, these results suggest that the D100R mutational effects on isolated RecA activities combine synergistically to perturb its higher-order functions. We conclude that the replacement of Asp100 resulted in a change in the electrostatic complementarity between RecA monomers during active filament assembly that prevents the protein from fully accessing the active multimeric state.
Links	PubMed Online version:10.1021/bi052296x
Keywords	Adenosine Diphosphate/analogs & derivatives; Adenosine Diphosphate/metabolism; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Aspartic Acid/physiology; DNA, Single-Stranded/metabolism; Models, Molecular; Nucleotides/metabolism; Protein Conformation; Rec A Recombinases/genetics; Rec A Recombinases/metabolism; Static Electricity; Substrate Specificity/drug effects; ortho-Aminobenzoates/metabolism
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

<annotationlinks/>

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.