Quantitation of the inhibition of Hfr x F- recombination by the mutagenesis complex UmuD'C

Abstract

The UmuD'C complex and RecA protein are two essential components in mutagenic repair of gaps produced by the replication of damaged DNA. In this process, the UmuD'C complex might help DNA polymerase to synthesize DNA across a lesion. Besides, a RecA polymer wrapping around single-stranded DNA could function as a directional chaperone to target the UmuD'C complex at the lesion. It was shown in our laboratory that the UmuD'C complex prevents homologous recombination and recombinational repair when expressed at elevated levels. To find out whether the UmuD'C complex inhibits recombination by interfering directly with RecA, we measured the kinetics of inhibition of Hfr x F- recombination in F- recipients in which either RecA or UmuD'C were made to vary. The cell concentrations of RecA and UmuD'C proteins were adjusted by having the recA and the umuD'C genes regulated by the arabinose P(BAD) promoter. In the absence of the UmuD'C complex, recombination was a function of RecA concentration and then reached a plateau when the RecA concentration was above 9000 monomers/cell. At a fixed RecA concentration, the yield of Hfr x F- recombinants decreased as a function of the UmuD'C cell concentration. At a given UmuD'C/RecA ratio, recombination inhibition by UmuD'C was reversed by increasing the RecA cell concentration. RecA1730, a mutant protein impaired in the chaperone activity, was insensitive to UmuD'C inhibition. We propose a model accounting for the RecA chaperone function in SOS mutagenesis and for the UmuD'C inhibitory effect on homologous recombination. We suggest that the UmuD'C complex is placed at the tip of a RecA polymer as a result of a treadmilling process. This would position the UmuD'C complex right at a lesion while the capping by UmuD'C would destabilize a RecA polymer and thereby abort the recombination process.