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RecA proteins from Deinococcus geothermalis and Deinococcus murrayi--cloning, purification and biochemical characterisation.

ABSTRACT: BACKGROUND: Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in ?-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown. RESULTS: In this study, recA genes from Deinococcus geothermalis and Deinococcus murrayi, bacteria that are slightly thermophilic and extremely ?-radiation resistant, were isolated, cloned and expressed in E. coli. After production and purification, the biochemical properties of DgeRecA and DmuRecA proteins were determined. Both proteins continued to exist in the solutions as heterogenous populations of oligomeric forms. The DNA binding by DgeRecA and DmuRecA proteins is stimulated by Mg2+ ions. Furthermore, both proteins bind more readily to ssDNA when ssDNA and dsDNA are in the same reaction mixture. Both proteins are slightly thermostable and were completely inactivated in 10 s at 80°C. Both proteins hydrolyze ATP and dATP in the presence of ssDNA or complementary ssDNA and dsDNA, but not in the absence of DNA or in the presence of dsDNA only, and dATP was hydrolyzed more rapidly than ATP. They were also able to promote DNA strand exchange reactions by a pathway common for other RecA proteins. However, we did not obtain DNA strand exchange products when reactions were performed on an inverse pathway, characteristic for RecA of D. radiodurans. CONCLUSIONS: The characterization of DgeRecA and DmuRecA proteins made in this study indicates that the unique properties of D. radiodurans RecA are probably not common among RecA proteins from Deinococcus sp.

SUBMITTER: Wanarska M

PROVIDER: S-EPMC3103430 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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RecA proteins from Deinococcus geothermalis and Deinococcus murrayi--cloning, purification and biochemical characterisation.

Wanarska Marta M Krawczyk Beata B Hildebrandt Piotr P Kur Józef J

BMC molecular biology 20110422

<h4>Background</h4>Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in γ-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown.<h4>Results</h4>In thi ...[more]

PMID: 21513512

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Project description:Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis.

Dataset Information

RecA proteins from Deinococcus geothermalis and Deinococcus murrayi--cloning, purification and biochemical characterisation.

Publications

RecA proteins from Deinococcus geothermalis and Deinococcus murrayi--cloning, purification and biochemical characterisation.

Similar Datasets

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