Abstract
When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.
Highlights
The biochemical data available for the Escherichia coli RecA protein and the reactions that it catalyzes (ATP hydrolysis and DNA strand exchange) are abundant [1, 7], but we are just
The RecA ATPase is a sensitive barometer of RecA filament status, and it has often been used as a real-time indicator of the level of RecA protein binding to DNA [3, 7, 11, 28, 30, 31]
At constant protein concentrations of RecA protein (1 M) and ssDNA (3 M), increasing the amount of DinI protein initially enhanced the rate of ATP hydrolysis by the wild-type RecA protein (Fig. 1)
Summary
The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response. RecA nucleates onto regions of single-stranded DNA (ssDNA) that develop as a result of DNA damage and forms a helical nucleoprotein filament This ATP-dependent activated RecA filament interacts with and promotes the autocatalytic cleavage of the LexA repressor protein, thereby inducing the genes of the SOS regulon [6]. More nearly stoichiometric concentrations, DinI has a very substantial stabilizing effect on RecA filaments and does not significantly inhibit many RecA functions. We further demonstrate that the RecA C terminus modulates the interaction between DinI and RecA
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