Discrimination of Rhizoma Gastrodiae (Tianma) using 3D synchronous fluorescence spectroscopy coupled with principal component analysis.

Rhizoma Gastrodiae (Tianma) of different variants and different geographical origins has vital difference in quality and physiological efficacy. This paper focused on the classification and identification of Tianma of six types (two variants from three different geographical origins) using three dimensional synchronous fluorescence spectroscopy (3D-SFS) coupled with principal component analysis (PCA). 3D-SF spectra of aqueous extracts, which were obtained from Tianma of the six types, were measured by a LS-50B luminescence spectrofluorometer. The experimental results showed that the characteristic fluorescent spectral regions of the 3D-SF spectra were similar, while the intensities of characteristic regions are different significantly. Coupled these differences in peak intensities with PCA, Tianma of six types could be discriminated successfully. In conclusion, 3D-SFS coupled with PCA, which has such advantages as effective, specific, rapid, non-polluting, has an edge for discrimination of the similar Chinese herbal medicine. And the proposed methodology is a useful tool to classify and identify Tianma of different variants and different geographical origins.

Knockouts of RecA-like proteins RadC1 and RadC2 have distinct responses to DNA damage agents in Sulfolobus islandicus.

RecA family recombinases play essential roles in maintaining genome integrity. A group of RecA-like proteins named RadC are present in all archaea, but their in vivo functions remain unclear. In this study, we performed phylogenetic and genetic analysis of two RadC proteins from Sulfolobus islandicus. RadC is closer to the KaiC lineage of cyanobacteria and proteobacteria than to the lineage of the recombinases (RecA, RadA, and Rad51) and the recombinase paralogs (e.g., RadB, Rad55, and Rad51B). Using the recently-established S. islandicus genetic system, we constructed deletion and over-expression strains of radC1 and radC2. Deletion of radC1 rendered the cells more sensitive to DNA damaging agents, methyl methanesulfonate (MMS), hydroxyurea (HU), and ultraviolet (UV) radiation, than the wild type, and a ΔradC1ΔradC2 double deletion strain was more sensitive to cisplatin and MMS than the ΔradC1 single deletion mutant. In addition, ectopic expression of His-tagged RadC1 revealed that RadC1 was co-purified with a putative structure-specific nuclease and ATPase, which is highly conserved in archaea. Our results indicate that both RadC1 and RadC2 are involved in DNA repair. RadC1 may play a general or primary role in DNA repair, while RadC2 plays a role in DNA repair in response to specific DNA damages.

Archaeal DNA helicase HerA interacts with Mre11 homologue and unwinds blunt-ended double-stranded DNA and recombination intermediates.

HerA is a novel family DNA helicases that exist ubiquitously in thermophilic archaea. The genes are linked to homologues of eukaryotic recombination and repair proteins Mre11 and Rad50 in some of the genomes. However, the relationship between HerA and the related proteins is unclear. In this study, a homologue from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHerA) was characterized and physical and functional interactions between StoHerA and StoMre11 (Mre11 from S. tokodaii) were studied. It was found that StoHerA was able to unwind blunt-ended double-stranded DNA (dsDNA), although with lower efficiency. StoHerA was also able to unwind Holliday junction, splayed-arm DNA, as well as 5'- or 3'-overhang with high efficiency. Pull-down and yeast two-hybrid analyses revealed that StoHerA interacted with StoMre11 physically. The helicase activity of StoHerA was stimulated by StoMre11, indicating a functional role of this interaction. In addition, site-directed mutagenesis of StoHerA was performed to analyze functions of conserved residues of StoHerA. Interestingly, mutation of E355 to alanine in Walker B resulted in not only loss of ATPase and DNA helicase activities, but also dsDNA-binding ability, indicating that this residue is involved in the coupling of ATP hydrolysis, dsDNA-binding, and helicase activities.