CDK targets Sae2 to control DNA-end resection and homologous recombination

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs), with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Delta null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.     

Phosphorylation of p27BBP/eIF6 and its association with the cytoskeleton are developmentally regulated in Xenopus oogenesis

p27^BBP/eIF6 is an evolutionarily conserved regulator of ribosomal function. It is necessary for 60S biogenesis and impedes improper joining of 40S and 60S subunits, regulated by protein kinase C or Efl1p. No data on p27^BBP/eIF6 during early development of Metazoa are available. We studied the distribution, post-translational changes and association with the cytoskeleton of p27^BBP/ eIF6 during Xenopus oogenesis and early development. Results indicate that p27^BBP/eIF6 is present throughout oogenesis, partly associated with 60S subunits, partly free and with little cytoskeleton bound. During prophase I, p27^BBP/eIF6 is detected as a single band of 27-kDa. Upon maturation induced by progesterone or protein kinase C, a serine-phosphorylated 29 kDa isoform appears and is kept throughout development to the neurula stage. Confocal microscopy showed that the distribution of p27^BBP/eIF6 and its association with the cytoskeleton varies according to oogenesis stages. Briefly, in stage 6 oocytes, p27^BBP/eIF6 has a limited dot-like distribution, and does not co-localize with cytokeratin, whereas upon maturation it spreads throughout the cytoplasm. After fertilization, a large fraction coalesces around cytomembranes and a cytochalasin B-sensitive co-localization with cytokeratin occurs. RNAse removes p27^BBP/eIF6 from the cytokeratin fibres. Developmental data suggest a role of p27^BBP/eIF6 in controlling ribosomal availability or regulating cross-talk between ribosomes and the cytoskeleton.Received 7 April 2005; received after revision 11 May 2005; accepted 25 May 2005R. Carotenuto and N. De Marco contributed equally to the paper     

The oxidation of cystamine and other sulfur-diamines by diamine-oxidase preparations

Riassunto Le diamine solforate cistamina e lantionamina sono ossidate con velocità comparabile a quella della cadaverina dalla diamino-ossidasi. Il solfone della lantionamina è ossidato più lentamente ed il disolfossido della cistamina non è ossidato affatto. Benchè dall'ossidazione della cistamina si produca una mole di ammoniaca per mole di substrato, il consumo di ossigeno è due volte più elevato del teorico, indicante una ulteriore ossidazione del prodotto metabolico della reazione. Tuttavia taurina, ipotaurina e solfati, non sono determinabili nel deproteinizzato finale.     

Structure and evolution of vertebrate aldehyde oxidases: from gene duplication to gene suppression

Aldehyde oxidases (AOXs) and xanthine dehydrogenases (XDHs) belong to the family of molybdo-flavoenzymes. Although AOXs are not identifiable in fungi, these enzymes are represented in certain protists and the majority of plants and vertebrates. The physiological functions and substrates of AOXs are unknown. Nevertheless, AOXs are major drug metabolizing enzymes, oxidizing a wide range of aromatic aldehydes and heterocyclic compounds of medical/toxicological importance. Using genome sequencing data, we predict the structures of AOX genes and pseudogenes, reconstructing their evolution. Fishes are the most primitive organisms with an AOX gene (AOXα), originating from the duplication of an ancestral XDH. Further evolution of fishes resulted in the duplication of AOXα into AOXβ and successive pseudogenization of AOXα. AOXβ is maintained in amphibians and it is the likely precursors of reptilian, avian, and mammalian AOX1. Amphibian AOXγ is a duplication of AOXβ and the likely ancestor of reptilian and avian AOX2, which, in turn, gave rise to mammalian AOX3L1. Subsequent gene duplications generated the two mammalian genes, AOX3 and AOX4. The evolution of mammalian AOX genes is dominated by pseudogenization and deletion events. Our analysis is relevant from a structural point of view, as it provides information on the residues characterizing the three domains of each mammalian AOX isoenzyme. We cloned the cDNAs encoding the AOX proteins of guinea pig and cynomolgus monkeys, two unique species as to the evolution of this enzyme family. We identify chimeric RNAs from the human AOX3 and AOX3L1 pseudogenes with potential to encode a novel microRNA.     

Synthesis and antitumour activity of new daunorubicin and adriamycin analogues

A new synthetic procedure for the preparation of daunorubicin and adriamycin analogues bearing different substituents on ring D, has been developed. The new compounds display outstanding efficacy against experimental tumours of mice.     

Observation of a pairing pseudogap in a two-dimensional Fermi gas

Pairing of fermions is ubiquitous in nature, underlying many phenomena. Examples include superconductivity, superfluidity of (3)He, the anomalous rotation of neutron stars, and the crossover between Bose-Einstein condensation of dimers and the BCS (Bardeen, Cooper and Schrieffer) regime in strongly interacting Fermi gases. When confined to two dimensions, interacting many-body systems show even more subtle effects, many of which are not understood at a fundamental level. Most striking is the (as yet unexplained) phenomenon of high-temperature superconductivity in copper oxides, which is intimately related to the two-dimensional geometry of the crystal structure. In particular, it is not understood how the many-body pairing is established at high temperature, and whether it precedes superconductivity. Here we report the observation of a many-body pairing gap above the superfluid transition temperature in a harmonically trapped, two-dimensional atomic Fermi gas in the regime of strong coupling. Our measurements of the spectral function of the gas are performed using momentum-resolved photoemission spectroscopy, analogous to angle-resolved photoemission spectroscopy in the solid state. Our observations mark a significant step in the emulation of layered two-dimensional strongly correlated superconductors using ultracold atomic gases.     

Theta-paced flickering between place-cell maps in the hippocampus

The ability to recall discrete memories is thought to depend on the formation of attractor states in recurrent neural networks. In such networks, representations can be reactivated reliably from subsets of the cues that were present when the memory was encoded, at the same time as interference from competing representations is minimized. Theoretical studies have pointed to the recurrent CA3 system of the hippocampus as a possible attractor network. Consistent with predictions from these studies, experiments have shown that place representations in CA3 and downstream CA1 tolerate small changes in the configuration of the environment but switch to uncorrelated representations when dissimilarities become larger. However, the kinetics supporting such network transitions, at the subsecond timescale, is poorly understood. Here we show in rats that instantaneous transformation of the spatial context does not change the hippocampal representation all at once but is followed by temporary bistability in the discharge activity of CA3 ensembles. Rather than sliding through a continuum of intermediate activity states, the CA3 network undergoes a short period of competitive flickering between preformed representations of the past and present environment before settling on the latter. Network flickers are extremely fast, often with complete replacement of the active ensemble from one theta cycle to the next. Within individual cycles, segregation is stronger towards the end, when firing starts to decline, pointing to the theta cycle as a temporal unit for expression of attractor states in the hippocampus. Repetition of pattern-completion processes across successive theta cycles may facilitate error correction and enhance discriminative power in the presence of weak and ambiguous input cues.