Joint Efforts of Replicative Helicase and SSB Ensure Inherent Replicative Tolerance of G-Quadruplex.

G-quadruplex (G4) is a four-stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single-molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging-strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single-stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading-strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

RPA transforms RNase H1 to a bidirectional exoribonuclease for processive RNA-DNA hybrid cleavage.

RNase H1 has been acknowledged as an endoribonuclease specializing in the internal degradation of the RNA moiety within RNA-DNA hybrids, and its ribonuclease activity is indispensable in multifaceted aspects of nucleic acid metabolism. However, the molecular mechanism underlying RNase H1-mediated hybrid cleavage remains inadequately elucidated. Herein, using single-molecule approaches, we probe the dynamics of the hybrid cleavage by Saccharomyces cerevisiae RNase H1. Remarkably, a single RNase H1 enzyme displays 3'-to-5' exoribonuclease activity. The directional RNA degradation proceeds processively and yet discretely, wherein unwinding approximately 6-bp hybrids as a prerequisite for two consecutive 3-nt RNA excisions limits the overall rate within each catalytic cycle. Moreover, Replication Protein A (RPA) reinforces RNase H1's 3'-to-5' nucleolytic rate and processivity and stimulates its 5'-to-3' exoribonuclease activity. This stimulation is primarily realized through the pre-separation of the hybrids and consequently transfers RNase H1 to a bidirectional exoribonuclease, further potentiating its cleavage efficiency. These findings unveil unprecedented characteristics of an RNase and provide a dynamic view of RPA-enhanced processive hybrid cleavage by RNase H1.

Optimizing carbon fibre supports for bioreactors by nitric acid oxidation and calcium ion coverage according to extended DLVO theory.

Optimizing supports for microorganisms is required for bioreactors. Carbon fibres (CF) were employed as supports for microorganisms. To optimize CF supports for immobilizing bacterial cells, we used methods of nitric acid oxidation and calcium ion coverage. We evaluated the capacity of these CF supports (untreated CF, nitric acid oxidation CF and Ca2+-covered CF) via bacterial cell adhesion tests, based on extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. The results implied that because of the high hamaker constants, oxidized CF supports had higher capacity in this regard than untreated CF supports. However, the growing oxygen groups increased the negative zeta potential of CF supports, thus likely to reduce their capacity, in accordance with XDLVO theory. Since the Ca2+ coverage could decrease the negative zeta potentials of CF without reducing the hamaker constants, it could enhance the capacity of oxidized CF supports. We concluded that a combination of nitric acid oxidation and Ca2+ coverage could increase the capacity of CF supports to immobilize bacterial cells.

Carbon nanotubes/carbon fiber hybrid material: a super support material for sludge biofilms.

Carbon fiber (CF) is widely used as a sludge biofilm support material for wastewater treatment. Carbon nanotubes/carbon fiber (CNTs/CF) hybrid material was prepared by ultrasonically assisted electrophoretic deposition (EPD). CF supports (CF without handling, CF oxidized by nitric acid, CNTs/CF hybrid material) were evaluated by sludge immobilization tests, bacterial cell adsorption tests and Derjaguin -Landau -Verwey -Overbeek (DLVO) theory. We found that the CNTs/CF hybrid material has a high capacity for adsorbing activated sludge, nitrifying bacterial sludge and pure strains (Escherichia coli and Staphylococcus aureus). CNTs deposited on CF surface easily wound around the curved surface of bacterial cell which resulted in capturing more bacterial cells. DLVO theory indicated the lowest total interaction energy of CNTs/CF hybrid material, which resulted in the highest bacteria cell adsorption velocity. Experiments and DLVO theory results proved that CNTs/CF hybrid material is a super support material for sludge biofilms.