A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism

The different steps of the human Top1 (topoisomerase I) catalytic cycle have been analysed in the presence of a pentacyclic-diquinoid synthetic compound. The experiments indicate that it efficiently inhibits the cleavage step of the enzyme reaction, fitting well into the catalytic site. Surprisingly the compound, when incubated with the binary topoisomerase–DNA cleaved complex, helps the enzyme to remove itself from the cleaved DNA and close the DNA gap, increasing the religation rate. The compound also induces the religation of the stalled enzyme–CPT (camptothecin)– DNA ternary complex. Analysis of the molecule docked over the binary complex, together with its chemical properties, suggests that the religation enhancement is due to the presence on the compound of two oxygen atoms that act as hydrogen acceptors. This property facilitates the deprotonation of the 5 DNA end, suggesting that this is the limiting step in the topoisomerase religation mechanism

Replacement of the human topoisomerase linker domain with the plasmodial counterpart renders the enzyme camptothecin resistant.

A human/plasmodial hybrid enzyme, generated by swapping the human topoisomerase IB linker domain with the corresponding domain of the Plasmodium falciparum enzyme, has been produced and characterized. The hybrid enzyme displays a relaxation activity comparable to the human enzyme, but it is characterized by a much faster religation rate. The hybrid enzyme is also camptothecin resistant. A 3D structure of the hybrid enzyme has been built and its structural-dynamical properties have been analyzed by molecular dynamics simulation. The analysis indicates that the swapped plasmodial linker samples a conformational space much larger than the corresponding domain in the human enzyme. The large linker conformational variability is then linked to important functional properties such as an increased religation rate and a low drug reactivity, demonstrating that the linker domain has a crucial role in the modulation of the topoisomerase IB activity

The human topoisomerase 1B Arg634Ala mutation results in camptothecin resistance and loss of inter-domain motion correlation.

Human topoisomerase 1B, the unique target of the natural anticancer compound camptothecin, catalyzes the unwinding of supercoiled DNA by introducing transient single strand nicks and providing covalent protein-DNA adducts. The functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to camptothecin as indicated by a viability assay of the yeast cells transformed with the mutated protein. The mutant also displays a very fast religation rate that is only partially reduced by the presence of the drug, suggesting that this is the main reason for its resistance. A comparative analysis of the structural-dynamical properties of the native and mutant proteins by molecular dynamics simulation indicates that mutation of Arg634 brings to a loss of motion correlation between the different domains and in particular between the linker and the C-terminal domain, containing the catalytic tyrosine residue. These results indicate that the loss of motion correlation and the drug resistance are two strongly correlated events

Stress response induced by carbon nanoparticles in Paracentrotus lividus

Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Despite the high rate of pollution generated by nano-pollutants, up to now their toxic effect on development is totally obscure. Embryos treated with carbon nanoparticles, RNA preparation, retro-transcription and quantitative real-time PCR. In response to carbon nano-particles exposure, the embryos collected 24 h later showed a 3,07-fold at 5x10(12) p and a 1,58-fold at 2.5x10(13) p and a 1,92-fold at 2.5x10(14) p increase in Pl14-3-3ε transcript levels compared with controls. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε. Pl14-3-3ε mRNA levels were measured by RT-PCR during development and found to increase from the mesenchyme blastula to the prism stage. Our results confirm the involvement of 14-3-3ε in the stress response elicited by carbon nano-particles

Specific detection of topoisomerase I from the malaria causing P. falciparum parasite using isothermal rolling circle amplification

We present a Rolling-Circle-Enhance-Enzyme-Activity-Detection (REEAD) system with potential use for future point-of-care diagnosis of malaria. In the developed setup, specific detection of malaria parasites in crude blood samples is facilitated by the conversion of single Plasmodium falciparum topoisomerase I (pfTopI) mediated cleavage-ligation events, happening within nanometer dimensions, to micrometer-sized products readily detectable at the single molecule level in a fluorescence microscope. In principle, REEAD requires no special equipment and the readout is adaptable to simple colorimetric detection systems. Moreover, with regard to detection limit the presented setup is likely to outcompete standard gold immuno-based diagnostics. Hence, we believe the presented assay forms the basis for a new generation of easy-to-use diagnostic tools suitable for the malaria epidemic areas in developing countries

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico

DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers.

Background DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects.MethodsUsing site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures.ResultsMutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity.ConclusionsIt can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons

Marine sediments remotely unveil long-term climatic variability over Northern Italy

A deep understanding of natural decadal variability is pivotal to discuss recently observed climate trends. Paleoclimate proxies allow reconstructing natural variations before the instrumental period. Typically, regional-scale reconstructions depend on factors like dating, multi-proxy weighting and calibration, which may lead to non-robust reconstructions. Riverine records inherently integrate information about regional climate variability, partly overcoming the above mentioned limitation. The Po River provides major freshwater input to Eastern Mediterranean, as its catchment encompasses a large part of Northern Italy. Here, using historical discharge data and oceanographic measurements, we show that Po River discharge undergo robust decadal fluctuations that reach the Ionian Sea, -1,000 km South of Po River delta, through propagating salinity anomalies. Based on this propagation, we use a high-resolution foraminiferal delta O-18 record from a sediment core in the Ionian Sea to reconstruct North Italian hydrological variability on millennial-scale for the first time. The reconstruction reveals highly significant decadal variability that persists over the last 2,000 years. Many reconstructed extremes correspond to documented catastrophic events. Our study provides the first millennial-scale reconstruction of the strength of decadal hydrological variability over Northern Italy. It paves the way to assess the persistence of large-scale circulation fingerprints on the North Italian climate