Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase

Aromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydrolysis reaction. The effect of the aromatic stacking on catalysis is peculiar in that uracil, the aromatic moiety influenced by the aromatic interaction is relatively distant from the site of hydrolysis at the alpha-phosphate group. Using crystallographic, kinetics, optical spectroscopy and thermodynamics calculation approaches we delineate a possible mechanism by which rate acceleration is achieved through the remote π–π interaction. The abundance of similarly positioned aromatic interactions in various nucleotide hydrolyzing enzymes (e.g. most families of ATPases) raises the possibility of the reported phenomenon being a general component of the enzymatic catalysis of phosphate ester hydrolysis.     

xni-deficient Escherichia coli are proficient for recombination and multiple pathways of repair

Single-strand-dependent DNA exonucleases play important roles in DNA repair and recombination in all organisms. In Escherichia coli the redundant functions provided by the RecJ, ExoI, ExoVII and ExoX exonucleases are required for mismatch repair, UV resistance and homologous recombination. We have examined whether the xni gene product, the single-strand exonuclease ExoIX, is also a member of this group. We find that deletion of xni has no effect on the above processes, or on resistance to oxidative damage, even in combination with other exonuclease mutations. We conclude that the xni gene product does not belong to this group of nucleases that play redundant roles in DNA recombination and repair.     

Stem cells: a view from the roots

In both plants and animals, regeneration requires the activation of stem cells. This is possibly related to the origin and requirements of multicellularity. Although long diverged from a common ancestry, plant and animal models such as Arabidopsis, Drosophila and mouse share considerable similarities in stem cell regulation. This includes stem cell niche organisation, epigenetic modification of DNA and histones, and the role of small RNA machinery in differentiation and pluripotency states. Dysregulation of any of these can lead to premature ageing, patterning and specification defects, as well as cancers. Moreover, emerging basal animal and plant systems are beginning to provide important clues concerning the diversity and evolutionary history of stem cell regulatory mechanisms in eukaryotes. This review provides a comparative framework, highlighting both the commonalities and differences among groups, which should promote the intelligent design of artificial stem cell systems, and thereby fuel the field of biomaterials science.     

Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.     

Long-term maintenance of Pinus nigra embryogenic cultures through cryopreservation

Embryogenic tissues of Pinus nigra have been cryopreserved using a two step slow-freezing method. In the first experiment, 20 cell lines were included and the effect of the duration of cryostorage (1 h vs. 1 year) on regrowth was compared. After a short-term storage (1 h in liquid nitrogen, LN) out of 20 cell lines tested 15 showed regrowth (75%) with individual frequencies 10–100%. Long term storage (1 year in LN) resulted in regrowth of 14 cell lines (70%) while the individual frequencies reached 10–100%. One year storage had no negative influence on the fresh mass accumulation evaluated 2–3 months after thawing. Another 20 cell lines were included in the second experiment with the aim to study the correlation between cryotolerance and maturation capacity of cell lines. Between maturation capacity and cryotolerance expressed as regrowth frequencies of individual cell lines, no correlation has been found.     

Construction of a Global Pain Systems Network Highlights Phospholipid Signaling as a Regulator of Heat Nociception

The ability to perceive noxious stimuli is critical for an animal''s survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.     

ABCC1 polymorphisms in anthracycline-induced cardiotoxicity in childhood acute lymphoblastic leukaemia

Anthracyclines are potent cytostatic drugs, the correct dosage being critical to avoid possible cardiac side effects. ABCC1 [ATP-binding cassette, sub-family C, member 1; also denoted as MRP1 (multidrug resistance-associated protein 1)] is expressed in the heart and takes part in the detoxification and protection of cells from the toxic effects of xenobiotics, including anthracyclines. Our objective was to search for associations between LV (left ventricular) function and single-nucleotide polymorphisms of the ABCC1 gene in children receiving anthracycline chemotherapy. Data of 235 paediatric patients with acute lymphoblastic leukaemia was analysed. Patients were followed-up by echocardiography (median follow-up 6.3 years). Nine polymorphisms in the ABCC1 gene were genotyped. The ABCC1 rs3743527TT genotype and rs3743527TT–rs246221TC/TT genotype combination were associated with lower LVFS (left ventricular fractional shortening) after chemotherapy. The results suggest that genetic variants in the ABCC1 gene influence anthracycline-induced LV dysfunction.     

Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with DYNAFIT

Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-alpha-thrombin, human activated protein C and human factor Xa were studied at pH 8.0-8.4 and 25.0+/-0.1 degrees C by the progress curve method and individual rate constants were calculated mostly within 10% internal error using DYNAFITV. A systematic strategy has been developed for fitting a three-step consecutive mechanism to eighteen hundred to six thousand time-course data points polled from two to four independent kinetic experiments. Enzyme and substrate concentrations were also calculated. Individual rate constants well reproduce published values obtained under comparable conditions and the Michaelis-Menten kinetic parameters calculated from these elementary rate constants are also within reasonable limits of published values. For comparison, the integrated Michaelis-Menten equation was also fitted to data from twelve sets. Both the k(cat) and k(cat)/K(m) values are within 15% agreement with those calculated using the elementary rate constants obtained with DYNAFITV. Rate constants for the second and third consecutive steps are within 3-4 fold indicating that both determine the overall rate. The Factor Xa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl at pH 8.4 in a series of buffers containing increasing fractions of deuterium at 25.0+/-0.1 degrees C shows a very strong dependence of k(3) and a moderate dependence of k(2) on D content in the buffer: the fractionation factors are: 0.49+/-0.03 for K(1,) 0.70+/-0.05 for k(2), and (0.32+/-0.03)(2) for k(3).