Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.
The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.
Ellouze
C
C
UMR 216, Centre Nastional de la Recherche Scientifique and Institut Curie, Orsay, France.
Selmane
T
T
Kim
H K
HK
Tuite
E
E
Nordén
B
B
Mortensen
K
K
Takahashi
M
M
D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009711:Nucleotides; D004247:DNA; D011938:Rec A Recombinases