Abstract
Binding of ATP to the RecA protein induces a high affinity DNA binding required for activation of enzyme function. Screens for in vivo recombination and repressor cleavage activities show Gln194 to be intolerant of all substitutions. Analyses of three mutant proteins (Q194N, Q194E, and Q194A) show that although basal enzyme function is maintained, each protein no longer displays an ATP-induced increase in DNA binding affinity. High salt activation of RecA function is also disrupted by these mutations. In contrast, ATP-induced changes in the oligomeric structure of RecA are maintained in the mutant proteins. These results demonstrate that Gln194 is a critical "allosteric switch" for ATP-induced activation of RecA function but is not the exclusive mediator of ATP-induced changes in RecA.
Highlights
The Escherichia coli RecA protein is a multifunctional enzyme that plays a central role in the processes of recombinational DNA repair, homologous genetic recombination, and the cellular SOS response to DNA damage [1,2,3]
The binding of RecA to ssDNA is regulated in a classic allosteric fashion, whereby the binding of ATP induces a high affinity DNA binding state of the protein [7, 8]
Mutations at Gln194 block the high salt activation of RecA function. These results indicate that Gln194 is an important “on-off” switch required for the general activation of RecA function
Summary
The Escherichia coli RecA protein is a multifunctional enzyme that plays a central role in the processes of recombinational DNA repair, homologous genetic recombination, and the cellular SOS response to DNA damage [1,2,3]. In the presence of ATP␥S, a dramatic increase is seen in the affinity of wild type RecA for ssDNA compared with the mutant proteins (Fig. 2B).
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