Halogenated acetic and acrylic acids from the red alga Asparagopsis taxiformis

Nine halogenated acetic acids and nine halogenated acrylic acids have been identified in the aqueous extract of Hawaiian Asparagopsis taxiformis.     

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.     

p53 stimulates human topoisomerase I activity by modulating its DNA binding

The tumor suppressor protein p53 and the human DNA topoisomerase I (htopoI) interact with each other, which leads to a stimulation of the catalytic activity of htopoI. Moreover, p53 stimulates the topoisomerase I-induced recombination repair (TIRR) reaction. However, little was known about how p53 stimulates this topoisomerase I activity. Here we demonstrate that monomeric p53 is sufficient for the stimulation of the topoisomerase I-catalyzed relaxation activity, but the tetrameric form of p53 is required for the stimulation of TIRR. We also show that p53 stimulates topoisomerase I activity by increasing the dissociation of htopoI from DNA. Since htopoI forms a closed ring structure around the DNA, our results suggest that p53 induces a conformational change within htopoI that results in an opening of the clamp, and thereby releases htopoI from DNA.     

Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation

Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase–loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3′-phosphotyrosine protein–DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre–loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a ‘cleavage-competent’ Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3′-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the ‘cleavage-competent’ subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the ‘non-cleaving’ subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer.     

Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase

The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA binding activity to nearly the same degree as wild-type human TS. RNA binding activity of TS:C43A was reduced by 30% when compared to wild-type TS, while TS:C180A was completely devoid of RNA binding activity. In vitro translation studies confirmed that mutant proteins TS:C43A, TS:C195A, TS:C199A and TS:C210A, significantly repressed human TS mRNA translation, while TS:C180A was unable to do so. To confirm the in vivo significance of the cysteine sulfhydryl residue, mutant proteins TS:C180A and TS:C195A were each expressed in human colon cancer HCT-C18:TS(–) cells that expressed a functionally inactive TS. A recombinant luciferase reporter gene under the control of a TS-response element was co-transfected into these same cells, and luciferase activity increased in the presence of the TS:C195A mutant TS protein to a level similar to that observed upon expression of wild-type TS protein. In contrast, luciferase activity remained unchanged in cells expressing the TS:C180A mutant protein. Taken together, these findings identify Cys-180 as a critical residue for the in vitro and in vivo translational regulatory effects of human TS.     

Characterisation of mutations in 77 patients with X-linked myotubular myopathy,including a family with a very mild phenotype

X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support. It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts ( S. cerevisiae and S. pombe). To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene. We have found additional mutations in 77 patients, including 35 novel ones. We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopathy, and in his two grandsons also mildly affected. Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations. With the present report, 192 different mutations in the MTM1 gene have been described in 328 families. The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years.     

Microtubule dynamics: new surprises from an old MAP

Dis1/XMAP215 family microtubule-binding proteins are essential for cell division in animals, plants and fungi, suggesting a conserved cell-division mechanism used by all eukaryotes. Two new studies, however, reveal that different family members can have very different effects on microtubule dynamics.