Giardia duodenalis Rad52 protein: biochemical characterization and response upon DNA damage.

Giardia duodenalis is a flagellated binucleated protozoan that colonizes the small intestine in mammals, causing giardiasis, acute or chronic diarrhea. DNA double strand break either endogenously or exogenously generated is a major insult to DNA and its repair by homologous recombination (HR) is crucial for genomic stability. During HR, Rad52 plays key roles in the loading of the Rad51 recombinase, and the annealing of the second double-strand break end to the displaced strand of the D-loop structure. Among the functions found in vitro in yeast and human Rad52 protein are: ssDNA or dsDNA binding activity, ability to anneal bare or RPA coated-ssDNA, as well as multimeric ring formation. In this work, we searched for conserved domains in a putative Rad52 protein from G. duodenalis (GdRad52). Its coding sequence was cloned, expressed and purified to study its biochemical properties. rGdRad52 binds to dsDNA and ssDNA, with greater affinity for the latter. Likewise, rGdRad52 promotes annealing of DNA uncoated and coated with GdRPA1. rGdRad52 interacts with GdDMC1B and with GdRPA1 protein as shown in far western blotting assay. Additionally, rGdRad52 formed multimeric rings as observed by electronic microscopy. Finally, GdRad52 is over expressed in response upon DNA damage inflicted on trophozoites.

Biochemical characterization of plant Rad52 protein from rice (Oryza sativa).

DNA damage in living cells is repaired by two main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Of all the genes promoting HR, Rad52 (Radiation sensitive 52) is an important gene which is found to be highly conserved across different species. It was believed that RAD52 is absent in plant systems until lately. However, recent genetic studies have shown the presence of RAD52 homologues in plants. Rad52 homologues in plant systems have not yet been characterized biochemically. In the current study, we bring out the biochemical properties of rice Rad52-2a protein. OsRad52-2a was over-expressed in Escherichia coli BL21 (DE3) cells and the protein was purified. The identity of purified OsRad52-2a protein was confirmed via peptide mass fingerprinting. Gel filtration and native PAGE analysis indicated that the OsRad52-2a protein in its native state probably formed an undecameric structure. Purified OsRad52-2a protein showed binding to single stranded DNA, double stranded DNA. Protein also mediated the renaturation of complementary single strands into duplex DNA in both agarose gel and FRET based assays. Put together, OsRad52-2a forms oligomeric structures and binds to ssDNA/dsDNA for mediating an important function like renaturation during homologous recombination. This study represents the first report on biochemical properties of OsRad52-2a protein from important crop like rice. This information will help in dissecting the recombination and repair machinery in plant systems.

Molecular anatomy of the recombination mediator function of Saccharomyces cerevisiae Rad52.

A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52 with Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function. Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNA-bound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad52 in the rad52 Delta327 background enhances the association of Rad51 protein with a HO-made DNA double-strand break and partially complements the methylmethane sulfonate sensitivity of the mutant cells. Our results provide a mechanistic framework for rationalizing the multi-faceted role of Rad52 in recombination and DNA repair.

Purification and assays of Saccharomyces cerevisiae homologous recombination proteins.

Homologous recombination is an important means of eliminating DNA double strand breaks from chromosomes. The homologous recombination reaction is mediated by the Rad51 recombinase, which requires a number of ancillary factors for maximal efficiency. The development of purification procedures and biochemical assays for yeast Rad51 and other yeast recombination proteins has allowed investigators to begin dissecting the hierarchy of physical and functional interactions among these protein factors that govern the integrity of the homologous recombination machinery. The biochemical studies done with yeast recombination factors have helped formulate conceptual frameworks to guide similar endeavors in other eukaryotes, including humans. Continuing efforts with reconstituted systems that comprise yeast factors will undoubtedly continue to provide insights into the mechanistic intricacy of the homologous recombination machinery.