A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA

Sequence conservation of schistosome cyclophilins

Sequence conservation of schistosome cyclophilin

Expression Cloning and Biochemical Characterizations of Recombinant Cyclophilin Proteins from Schistosoma mansoni

Recombinant Schistosoma mansoni cyclophilin proteins of the A and the B subtypes (SmCYP A and B) were expressed in bacterial cells as histidine- and maltose-binding fusion proteins and also as nonfused proteins. In addition, S. mansoni CYPs were produced in Sf9 insect cells in their natural forms. Purified recombinant SmCYP B was found to possess a peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 8.2 x 10(5) M-1 s-1. The SmCYP B isoform is approximately two to three times more active than SmCYP A. SmCYP B-specific RNA appears to be more abundant in adult schistosomes than SmCYP A RNA in Northern blots. These results support the conclusion that SmCYP B represents the major schistosomal CYP. The PPIase-associated activity of both CYPs was inhibitable by the immunosuppressive drug cyclosporin A (CsA). We attempt to explain differences in PPIase activities and in CsA inhibition by examining models of the two CYPs complexed to Cs

Application of synthetic peptides in development of a serologic method for laboratory diagnosis of schistosomiasis mansoni

The immunoreactivity of seven peptides synthesized from Schistosoma mansoni proteins, was evaluated by dot-blot and ELISA assays using two different sensitization methodologies. The best results were obtained on wells of the Costar 3590 microplates coated with peptides P1, P2, P3, P6, and P7 using conventional methodology. The signals increased considerably (p < 0.0003) on wells sensitized with P1 to P6 using alternative methodology. In contrast, the well coated with peptide P7 presented lower signal when compared with conventional methodology (p = 0.0019). These results, establish the basis for the application of synthetic peptides for laboratory diagnosis of schistosomiasis mansoni