Structure and Properties of DNA Molecules Over The Full Range of Biologically Relevant Supercoiling States

Topology affects physical and biological properties of DNA and impacts fundamental cellular processes, such as gene expression, genome replication, chromosome structure and segregation. In all organisms DNA topology is carefully modulated and the supercoiling degree of defined genome regions may change according to physiological and environmental conditions. Elucidation of structural properties of DNA molecules with different topology may thus help to better understand genome functions. Whereas a number of structural studies have been published on highly negatively supercoiled DNA molecules, only preliminary observations of highly positively supercoiled are available, and a description of DNA structural properties over the full range of supercoiling degree is lacking. Atomic Force Microscopy (AFM) is a powerful tool to study DNA structure at single molecule level. We here report a comprehensive analysis by AFM of DNA plasmid molecules with defined supercoiling degree, covering the full spectrum of biologically relevant topologies, under different observation conditions. Our data, supported by statistical and biochemical analyses, revealed striking differences in the behavior of positive and negative plasmid molecules

First thermostable CLIP-tag by rational design applied to an archaeal O6-alkyl-guanine-DNA-alkyl-transferase

Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O6-benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC8. The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC8 was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O6-alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag variant

Su di in caso di lesioni da elica conseguenti al naufragio di un motopeschereccio

Gli Autori descrivono un caso relativo al naufragio di un motopeschereccio, infrantosi contro gli scogli dell’isola di Capraia, con reperimento del cadavere dell’unico soggetto a bordo, che presentava estese mutilazioni e plurimi complessi fratturativi, senza segni di vitalità. La ricostruzione della vicenda, condotta collegialmente tra medici legali ed un ingegnere navale, consentiva di ipotizzare che il pescatore fosse rimasto vittima di un malore poco dopo la partenza e che, successivamente al naufragio, il corpo, oltre che agli effetti del violento impatto contro gli scogli, fosse stato sottoposto all’azione lesiva combinata dell’elica del natante e delle cime rimaste avvolte nelle pale dell’elica stessa

Restoration of the activity of active-site mutants of the hyperthermophilic beta-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles

The beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of the reaction, and Glu206, which acts as the general acid/base catalyst. The activities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly were restored by externally added nucleophiles. Sodium azide and sodium formate were used as external nucleophiles and the products of their reaction were characterized. Glu387Ala/Gly mutants were reactivated with 2, 4-DNP-beta-Glc substrate and the Glu387Gly mutant showed recovered activity, with the same nucleophiles, also on 2-NP-beta-Glc. The reaction catalyzed by the Glu387Gly mutant proceeded differently depending on the type of externally added nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, thereby yielding an inverting glycosidase. Sodium formate promoted the opposite behavior (retaining) in the mutant, producing 3-O-beta-linked disaccharide derivative of the substrates. A possible role of sodium formate as a biomimicking agent in replacing the natural nucleophile Glu387 is also discussed

Reverse gyrase and genome stability in hyperthermophilic organisms

Reverse gyrase is a DNA topoisomerase that is peculiar in many aspects: it has the unique ability to introduce positive supercoils into DNA molecules; it comprises a type IA topoisomerase fused to a helicase-like domain; although it is a type IA topoisomerase, its reaction is ATP-dependent; and it is the only hyperthermophile-specific protein. All these features have made reverse gyrase the subject of biochemical, structural and functional studies, although they have not shed complete light on the evolution, mechanism and function of this distinctive enzyme. In the present article, we review the latest progress on structure-function relationships of reverse gyrase, and discuss old and recent data linking reverse gyrase to DNA stability, protection and repair in hyperthermophilic organisms