Binding of the recA gene product from E. coli to nucleic acids

196 1.5.3 Cazenave, C., M. Chabbert, J.J. Toulme and C. Helene, Laboratoire de Biophysique, Musrum National d'Histoire Naturelle, INSERM U.201, ERA CNRS 951, 61, Rue Buffon, 75005 Paris (France) Binding of the recA gene product from E. coli to nucleic acids The recA gene product from E. coli is involved in two major processes: general genetic recombination and DNA repair. Both require binding of nucleic acids and ATP. At low ionic strength, the RecA protein preferentially binds to double-stranded DNA as shown by an increase in melting temperature and by competition experiments. At moderate ionic strengths and in the presence of Mg 2÷ ions, RecA protein binding is stronger to single strands. ATP (or ATPTS) binds to RecA protein and first dissociates the RecA-ssDNA complex. Then slow binding takes place. Hydrolysis of ATP finally leads to a release of the R e c A - A D P complex from its ssDNA complex. The following scheme summarizes our observations: RecA protein binds to single-stranded DNA (complex I). ATP (or ATPyS) dissociates the complex while forming recA-ATP. If Mg 2÷ is present, the ternary complex ( r e c A - A T P - M g 2÷) binds to the single-stranded DNA via a slow nucleation-elongation process, reminiscent of the polymerization of actin in the presence of the ATP. The polymeric [ r e c A - A T P - M g 2+ ] n - D N A complex which is formed (complex II) is structurally different from complex I. Then upon hydrolysis of ATP, recA protein dissociates from the polynucleotide due to the low affinity of the r e c A - A D P - M g 2÷ complex for the nucleic acid.

1.5.4 Chanet, R., and C. Cassier, Institut Curie-Biologie, Centre Universitaire, Bht. 110, 91405 Orsay (France) Lethal and genotoxic effects of different photoproducts induced by psoralen derivatives using a double irradiation protocol in wild-type and in excision-defective yeast strains. Evidence for a new type of lesion Treatment of cells with 8-methoxypsoralen (8-MOP) followed by irradiation with 365-nm light (UVA) produces monoadducts and interstrand DNA crosslinks. If a second high dose of UVA is given to cells pretreated with 8-MOP + UVA, in the absence of 8-MOP, a fraction of initially induced monoadducts is transformed into crosslinks. We show that crosslinks are premutagenic and prerecombinogenic lesions in repair proficient Saccharomyces cerevisiae. The repair of crosslinks requires the excision-repair mechanism. Crosslinks appear to lead essentially to lethal events in excision-defective strains whereas monoadducts are tolerated. Monoadditions are only slightly mutagenic in such strains. We show that a double irradiation is mutagenic after 8-MOP treatment in the excision-defective strains. The leakiness of the strains is excluded by examining a mutant containing a disrupted rad gene. On the other hand a mutagenic effect is produced by a second UVA dose when an excision-defective strain is treated with a monofunctional derivative, 3-carbethoxypsoralen (3-CPs) plus UVA. This implies that new lesions are induced by the second UVA dose after pretreatment with a monofunctional agent. In order to reconcile the findings that: (a) crosslinks are essentially lethal lesions in excision-defective strains and that (b) a double irradiation after 8-MOP treatment is mutagenic in such strains, we suggest that similarly to the case of 3-CPs, new lesions may be involved in the mutagenic effect observed after irradiation with a second UVA dose also following treatment with 8-MOP. If however the effect of the double irradiation with monofunctional agents turns out to be limited to