Pol kappa: A DNA polymerase required for sister chromatid cohesion

Establishment of cohesion between sister chromatids is coupled to replication fork passage through an unknown mechanism. Here we report that TRF4, an evolutionarily conserved gene necessary for chromosome segregation, encodes a DNA polymerase with beta-polymerase-like properties. A double mutant in the redundant homologs, TRF4 and TRF5, is unable to complete S phase, whereas a trf4 single mutant completes a presumably defective S phase that results in a failure of cohesion between the replicated sister chromatids. This suggests that TRFs are a key link in the coordination between DNA replication and sister chromatid cohesion. Trf4 and Trf5 represent the fourth class of essential nuclear DNA polymerases (designated DNA polymerase kappa) in Saccharomyces cerevisiae and probably in all eukaryotes.     

DNA polymerase required for rapid repair of x-ray--induced DNA strand breaks in vivo

A much higher yield of DNA single-strand breaks was obtained in the DNA polymerase-deficient mutant Escherichia coli K-12 pol A1 after a given dose of x-rays than had been found before in Escherichia coli. The increased yield of single-strand breaks was due to the absence of a rapid repair system, which had not been described in Escherichia coli K-12. This absence probably accounts for the x-ray sensitivity of the pol A1 mutant. The rapid repair system can be reversibly inhibited in pol+ cells.     

Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions

Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair. Here, we demonstrate that the Drosophila Tip60 (dTip60) chromatin-remodeling complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. Both the histone acetyltransferase dTip60 as well as the adenosine triphosphatase Domino/p400 catalyze the exchange of phospho-H2Av. Thus, these data reveal a previously unknown mechanism for selective histone exchange that uses the concerted action of two distinct chromatin-remodeling enzymes within the same multiprotein complex.     

PDGF-induced activation of phospholipase C is not required for induction of DNA synthesis

Platelet-derived growth factor (PDGF) induction of DNA synthesis is believed to involve activation of phospholipase C (PLC) and subsequent accumulation of inositol 1,4,5-triphosphate [I(1,4,5)P3], increase in intracellular Ca2+, activation of protein kinase C (PKC), and receptor down regulation. Generation of these events is triggered by the tyrosine protein kinase (TPK) activity of the PDGF receptor. The TPK inhibitor genistein blocked PDGF induction of these events, including DNA synthesis, with the exception of receptor down regulation. PDGF-induced phosphotyrosine phosphorylations, including receptor autophosphorylation, were inhibited by genistein. Removal of genistein and PDGF resulted in DNA synthesis without the occurrence of PLC activation. These findings indicate that these early events, with the exception of receptor down regulation, are not necessary for PDGF-induced DNA synthesis.     

Early tagging of cortical networks is required for the formation of enduring associative memory

Although formation and stabilization of long-lasting associative memories are thought to require time-dependent coordinated hippocampal-cortical interactions, the underlying mechanisms remain unclear. Here, we present evidence that neurons in the rat cortex must undergo a "tagging process" upon encoding to ensure the progressive hippocampal-driven rewiring of cortical networks that support remote memory storage. This process was AMPA- and N-methyl-D-aspartate receptor-dependent, information-specific, and capable of modulating remote memory persistence by affecting the temporal dynamics of hippocampal-cortical interactions. Post-learning reinforcement of the tagging process via time-limited epigenetic modifications resulted in improved remote memory retrieval. Thus, early tagging of cortical networks is a crucial neurobiological process for remote memory formation whose functional properties fit the requirements imposed by the extended time scale of systems-level memory consolidation.     

A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA

A small RNA of Bacillus subtilis bacteriophage phi 29 is shown to have a novel and essential role in viral DNA packaging in vitro. This requirement for RNA in the encapsidation of viral DNA provides a new dimension of complexity to the attendant protein-DNA interactions. The RNA is a constituent of the viral precursor shell of the DNA-packaging machine but is not a component of the mature virion. Studies of the sequential interactions involving this RNA molecule are likely to provide new insight into the structural and possible catalytic roles of small RNA molecules. The phi 29 assembly in extracts and phi 29 DNA packaging in the defined in vitro system were strongly inhibited by treatment with the ribonucleases A or T1. However, phage assembly occurred normally in the presence of ribonuclease A that had been treated with a ribonuclease inhibitor. An RNA of approximately 120 nucleotides co-purified with the phi 29 precursor protein shell (prohead), and this particle was the target of ribonuclease action. Removal of RNA from the prohead by ribonuclease rendered it inactive for DNA packaging. By RNA-DNA hybridization analysis, the RNA was shown to originate from a viral DNA segment very near the left end of the genome, the end packaged first during in vitro assembly.     

Potentiation of bleomycin lethality by anticalmodulin drugs: a role for calmodulin in DNA repair

Treatment of exponentially growing Chinese hamster ovary cells with bleomycin causes a dose-dependent decrease in cell survival due to DNA damage. This lethal effect can be potentiated by the addition of a nonlethal dose of the anticalmodulin drug N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide ( W13 ) but not its inactive analog N-(4-aminobutyl)-2-naphthalenesulfonamide ( W12 ). By preventing the repair of damaged DNA, W13 also inhibits recovery from potentially lethal damage induced by bleomycin. These data suggest a role for calmodulin in the DNA repair pathway.     

A mitotic lamin B matrix induced by RanGTP required for spindle assembly

Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate-bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.     

Second structural motif for recognition of DNA by oligonucleotide-directed triple-helix formation

Relative orientations of the DNA strands within a purine.purine.pyrimidine triple helix have been determined by affinity cleaving. A purine-rich oligonucleotide bound in the major groove of double-helical DNA antiparallel to the Watson-Crick purine strand. Binding depended upon the concentration of multivalent cations such as spermine or Mg2+, and appeared to be relatively independent of pH. Two models with specific hydrogen-bonding patterns for base triplets (G.GC, A.AT, and T.AT) are proposed to explain the sequence specificity of binding. The two models differ in the conformation about the glycosyl bond (syn or anti) and the location of the phosphate-deoxyribose backbone in the major groove of DNA. This motif broadens the structural frameworks available as a basis for the design of sequence-specific DNA binding molecules.     

BRITTLE CULM16(BRITTLE NODE) is required for the formation of secondary cell walls in rice nodes

Plant cell walls constitute the skeletal structures of plant bodies,and thus confer lodging resistance for grain crops.While the basic cell wall synthesis machinery is relatively well established now,our understanding of how the process is regulated remains limited and fragmented.In this study,we report the identification and characterization of the novel rice(Oryza sativa L.)brittle culm16(brittle node;bc16)mutant.The brittle node phenotype of the bc16 mutant appears exclusively at nodes,and resembles the previously reported bc5 mutant.Combined histochemical staining and electron microscopy assays revealed that in the bc16 mutant,the secondary cell wall formation and thickening of node sclerenchyma tissues are seriously affected after heading.Furthermore,cell wall composition assays revealed that the bc16 mutation led to a significant reduction in cellulose and lignin contents.Using a map-based cloning approach,the bc16 locus is mapped to an approximately 1.7-Mb region of chromosome 4.Together,our findings strengthen evidence for discretely spatial differences in the secondary cell wall formation within plant bodies.     

Pattern formation in homogeneous polymer solutions induced by a continuous-wave visible laser

We report an unexpected nonphotothermal material organization induced by continuous-wave visible laser light at low power levels. This effect is observed along the laser beam propagation direction in fully transparent entangled solutions of common homopolymers featuring sufficiently high molecular mass and optical anisotropy along the chain backbone. The resulting formation of long-lived stringlike or dotlike patterns on the micrometer scale, probed by dark-field coherent imaging, depends on the molecular mass, architecture, solvent nature, and polymer concentration. Electrostrictive and alignment forces as well as chain cooperativity are responsible for the osmotic compression of the polymer solute. Subsequent waveguiding effects induce autoamplification and "pattern writing" upon prolonged illumination. This wave-medium coupling could potentially lead to photorefractive, microoptics, and nanotechnology applications.     

Phosphatidylserine receptor is required for clearance of apoptotic cells

Cells undergoing apoptosis during development are removed by phagocytes, but the underlying mechanisms of this process are not fully understood. Phagocytes lacking the phosphatidylserine receptor (PSR) were defective in removing apoptotic cells. Consequently, in PSR-deficient mice, dead cells accumulated in the lung and brain, causing abnormal development and leading to neonatal lethality. A fraction of PSR knockout mice manifested a hyperplasic brain phenotype resembling that of mice deficient in the cell death-associated genes encoding Apaf-1, caspase-3, and caspase-9, which suggests that phagocytes may also be involved in promoting apoptosis. These data demonstrate a critical role for PSR in early stages of mammalian organogenesis and suggest that this receptor may be involved in respiratory distress syndromes and congenital brain malformations.     

Autoradiographic plaques for the detection of antibody formation to soluble proteins by single cells

Single cells that synthesize antibody to soluble proteins can be detected and counted by an autoradiographic procedure. The method is based on the specific binding of I(131)-labeled antigen to the antidogy which has been produced by and has diffused around cells in an agarose medium and which has been precipitated by an antibody to gamma globulin. The antigen-binding zones appear as dark spots on high-speed x-ray film exposed to the dried agarose preparations.     

Tetraplex formation of a guanine-containing nonameric DNA fragment

A combination of spectroscopic and calorimetric techniques has been used to characterize the structures formed by a family of short, guanine-containing DNA single strands of the form d[GGTTXTTGG], X = A, C, G, T. In 1 molar NaCl at low temperatures, these molecules do not behave like single strands, but rather exhibit properties consistent with tetraplex formation. The standard state enthalpies, entropies, and free energies for formation of each tetraplex have been measured, as have preliminary nuclear magnetic resonance (NMR) spectra. In 1 molar KCl, the melting behavior of the structure or structures is more complex than in 1 molar NaCl. This observation may be related to the recently proposed "sodium-potassium switch."     

Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response

The BRCT repeats of the breast and ovarian cancer predisposition protein BRCA1 are essential for tumor suppression. Phosphopeptide affinity proteomic analysis identified a protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-Ser-X-X-Phe motif. Abraxas binds BRCA1 to the mutual exclusion of BACH1 (BRCA1-associated C-terminal helicase) and CtIP (CtBP-interacting protein), forming a third type of BRCA1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM)-containing protein RAP80 to BRCA1. Both Abraxas and RAP80 were required for DNA damage resistance, G(2)-M checkpoint control, and DNA repair. RAP80 was required for optimal accumulation of BRCA1 on damaged DNA (foci) in response to ionizing radiation, and the UIM domains alone were capable of foci formation. The RAP80-Abraxas complex may help recruit BRCA1 to DNA damage sites in part through recognition of ubiquitinated proteins.