The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.
Lichens, are symbiotic organisms of fungal and an algal and/or cyanobacterial partner and are considered edible or used for their medicinal properties by many cultures. They produce a variety of secondary compounds unique to lichens, mostly from fungal metabolism. In spite of the diversity few studies have evaluated the biological activity of secondary metabolites from lichens. Our objective was to evaluate the antioxidant, antimicrobial and anti‐adhesive activity of common lichens in Central Texas against Pseudomonas aeruginosa. Lichens were harvested from trees and rocks using standard techniques and identified by morphological, biochemical and microscopic analysis. The low molecular weight fraction (LMWF) and high molecular weight fraction (HMWF) were isolated using standard protocols. Antioxidant activity was determined by Malondialdehyde (MDA) assay and radical quenching assay. Antimicrobial and anti‐adhesive activity of sterile extracts against P. aeruginosa was determined by Kirby‐Bauer (KB) and ex vivo skin assay respectively. Lichens harvested were identified as Ramalina celastri (RA), R. stenospora (RS), R. Americana (RA), T. chrysopthalmus (TC), Parmotrema austrosinese (PA) and P. perforatum (PP). The HMWF had higher antioxidant activity and neutralized 60‐75% radicals compared to LMWF which only neutralized 20‐40% of the radicals except PA whose activity was 79%. All extracts were able to reduce MDA formation by 30‐88% except LMWF of RA and PP. None of the extracts had antimicrobial activity in the KB assay. All the HMWF were able to reduce adhesion of P. aeruginosa by 30‐55% except PP. LMWF from RS, PP, PA and PP reduced bacterial adhesion by 41‐54%. Our results suggest that Lichens can be an important source for bioactive compounds with benefits against oxidative and infectious diseases and warrants further investigation.
Simian Virus 40 (SV40) is a double stranded DNA virus helpful as a model system for eukaryotic cellular DNA replication. It encodes a Large T‐antigen (T‐ag) protein, which is essential for viral genome replication and regulation of the host cell cycle. Acting as a helicase enzyme, T‐ag is able to unwind various forms of DNA. Guanine rich sequences of DNA capable of forming G‐quadruplex knot‐like structures have been identified in several eukaryotic oncogene promoter regions, as well as in the SV40 virus genome, and may possibly influence transcription activity. T‐ag forms an active hexameric unit in the presence of ATP, and was demonstrated to bind single‐strand, duplex, and G‐quadruplex DNA using real time surface plasmon resonance (SPR). Probing with single strand binding protein (SSB) confirmed that T‐ag activity resulted in the unwinding of an intermolecular quadruplex.
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