Secondary Structure of RecA in Solution - The Effects of Cofactor, DNA and Ionic Conditions
Artikel i vetenskaplig tidskrift, 1995

The interactions of RecA with double-stranded DNA and with cofactor adenosine 5'-[3-thio]triphosphate (ATP[S] an analog of ATP) have been characterized by circular dichroism (CD) spectroscopy in a search for conformational changes associated with the formation of helical RecA ATP . DNA . fibers. Upon interaction with the RecA protein the cofactor is found to be structurally perturbed, possibly towards the syn ribose form of ATP[S], while the secondary structure of RecA remains unaffected. By contrast, when the ATP[S] . RecA . DNA complex is formed, a distinct change of the protein CD spectrum near 200 nm is observed as a result of interaction of RecA with DNA. The main change occurs upon the binding of the first DNA molecule [RecA can bind up to three DNA molecules simultaneously; Takahashi, M., Kubista, M. and Norden, B. (1991) Biochimie (Paris) 73, 219-226] and the effect appears to be independent of DNA sequence, suggesting a general change of protein conformation upon DNA binding. The CD of DNA is changed, indicating an alteration of the DNA structure, possibly related to stretching and unwinding. A small, reversible, decrease in the CD amplitude of RecA was observed when raising the temperature from 4 degrees C to 30 degrees C. The CD of RecA increases slightly with pH (up to 7.8) but is constant between pH 6.0 and 6.8. At pH below 6.0 or higher temperature (40 degrees C) slow irreversible denaturation of RecA occurs. The CD signal is effectively independent of salt, even in 2.2 M NaCl or 1 M sodium acetate, which is relevant regarding reported ATPase and coprotease activities promoted by salt. For high concentrations of magnesium (10 mM) at 30 degrees C the CD of RecA changes markedly and the appearance of light scattering indicates aggregation.

filaments

escherichia-coli

spectroscopy

circular dichroism

protein-binding

recombination

double-stranded dna

reca protein

duplex dna

activation

conformation

protein dna complex

linear dichroism

complexes

neutron-scattering

Författare

Pernilla Wittung

Institutionen för fysikalisk kemi

Bengt Nordén

Institutionen för fysikalisk kemi

M. Takahashi

European Journal of Biochemistry

0014-2956 (ISSN)

Vol. 228 1 149-154

Ämneskategorier

Biokemi och molekylärbiologi

DOI

10.1111/j.1432-1033.1995.0149o.x

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2018-10-15