Accessibility to modification of histidine residues of RecA protein upon DNA and cofactor binding.
The potential role of histidine residues of RecA protein in binding DNA has been investigated by monitoring their accessibility to diethylpyrocarbonate. In the absence of both DNA and cofactor, only one of two histidine residues is modified by the reagent, indicating that the other residue is buried. However, both histidine residues become accessible after addition of cofactor analog adenosine 5'-O-(3-thiotriphosphate) (ATP[S]) indicating a change in the organization of the RecA filament and/or a change in the conformation of protein. The diethylpyrocarbonate-modified RecA is found to be able to polymerize just as the unmodified protein. The binding of double-stranded DNA, in the presence of ATP[S], reduces the reactivity of both histidine residues to diethylpyrocarbonate. The binding of single-stranded DNA (with ATP[S]) has a similar, though smaller, protective effect. However, no significant dissociation of either of the complexes as a result of the modification was observed and a RecA molecule which had been modified in the absence of DNA could still bind DNA. A protection of the histidine residues is also effected by high salt concentration which promotes, just as DNA binding, ATPase and coprotease activity in RecA. The protection of histidine residues to diethylpyrocarbonate upon DNA binding probably relates to a conformational change of RecA and may not be any direct effect of shielding by the DNA. Nonetheless, the domains including the histidine residues could be centers of allosteric effects and are concluded to be close to the DNA binding site.
Takahashi
M
M
URA 1342 CNRS, Institut Curie, Université Paris Sud, Orsay, France.
Nordén
B
B
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*
D004277:DNA, Single-Stranded; C022571:adenosine 5'-O-(3-thiotriphosphate); D006639:Histidine; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases; D004047:Diethyl Pyrocarbonate