Search

To evaluate the importance of RecA in DNA double-strand break (DSB) repair, we examined the effect of low and high RecA concentrations such as 2500 and 100 000 molecules per cell expressed from the inducible Pspac promoter in Deinococcus radiodurans in absence or in presence of IPTG respectively. We showed that at low concentration, RecA has a negligible effect on cell survival after gamma-irradiation when bacteria were immediately plated on TGY agar whereas it significantly decreased the survival to gamma-irradiation of DeltaddrA cells while overexpression of RecA can partially compensate the loss of DdrA protein. In contrast, when cells expressing limited concentration of RecA were allowed to recover in TGY2X liquid medium, they showed a delay in mending DSB, failed to reinitiate DNA replication and were committed to die during incubation. A deletion of irrE resulted in sensitivity to gamma-irradiation and mitomycin C treatment. Interestingly, constitutive high expression of RecA compensates partially the DeltairrE sensitization to mitomycin C. The cells with low RecA content also failed to cleave LexA after DNA damage. However, neither a deletion of the lexA gene nor the expression of a non-cleavable LexA(Ind-) mutant protein had an effect on survival or kinetics of DNA DSB repair compared with their lexA+ counterparts in recA+ as well as in bacteria expressing limiting concentration of RecA, suggesting an absence of relationship between the absence of LexA cleavage and the loss of viability or the delay in the kinetics of DSB repair. Thus, LexA protein seems to play no major role in the recovery processes after gamma-irradiation in D. radiodurans.

Analysis of the complete genome indicates that insertion sequences (ISs) are abundant in the radio-resistant bacterium Deinococcus radiodurans. By developing a forward mutagenesis assay to detect any inactivation events in D. radiodurans, we found that in the presence of an active mismatch repair system 75% of the mutations to trimethoprim-resistance (Tmp(R)) resulted from an IS insertion into the thyA coding region. Analysis of their distribution among the spontaneous Tmp(R) mutants indicated that five different ISs were transpositionally active. A type II Miniature Inverted-repeat Transposable Element (MITE), related to one of the deinococcal ISs, was also discovered as an insertion into thyA. Seven additional genomic copies of this MITE element were identified by BLASTN. Gamma-ray irradiation of D. radiodurans led to an increase of up to 10-fold in the frequency of Tmp(R) mutants. Analysis of the induced mutations in cells exposed to 10 kGy indicated that gamma-irradiation induced transposition of ISDra2 approximately 100-fold. A 50-fold induction of ISDra2 transposition was also observed in cells exposed to 600 J m(-2) UV-irradiation. Point mutations to rifampicin resistance (Rif(R)) were also induced by gamma-irradiation to reach a plateau at 2 kGy. The plateau value represented a 16-fold increase in the mutant frequency over the background. Although error-free repair strategies predominate in D. radiodurans, an upregulation of transposition, as well as induction of point mutations in cells recovering from DNA damage, provide a genetic variability that may have long-term evolutionary consequences on the fitness of this organism in its habitat.

In addition to being an essential component of trans-lesion synthesis, the UmuD'C complex is an antagonist of RecA-mediated homologous recombination. When constitutively expressed at an elevated concentration, the UmuD'C complex sensitizes recA+ bacteria to DNA damage, whereas it has no effect on bacteria expressing a RecA [UmuR] protein that overcomes recombination inhibition. Using as a genetic screen enhanced cell killing on mitomycin plates, we isolated novel umuD' and umuC mutations that restored mitomycin sensitivity to recA D112G [UmuR] bacteria overproducing the UmuD'C complex. The mutations were named [Rin++] because a characterization in a recA+ as well in a recA D112G background showed that they enhanced UmuD'C-promoted recombination inhibition in two assays, conjugational recombination and recombinational repair of palindrome-containing DNA. The [Rin++] mutations affect five amino acids, G25D, S28T, P29L, E35K, and T95R, in UmuD' and seven, F10L, Y270C, K277E, F287L, F287S, K342Q and F351I, in UmuC. These amino acids might play a key role in the UmuD'C anti-recombination activity. None of the [Rin++] mutations enhanced UmuD'C-promoted mutagenic bypass of UV lesions, in contrast, several lead to a defect in this process. In this study, we discuss a few molecular mechanisms that could account for the recombination and mutagenesis phenotypes of a mutant UmuD'C [Rin++] complex.