Resistance to therapy caused by intragenic deletion in BRCA2

Cells with loss of BRCA2 function are defective in homologous recombination (HR) and are highly sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP), which provides the basis for a new therapeutic approach. Here we show that resistance to PARP inhibition can be acquired by deletion of a mutation in BRCA2. We derived PARP-inhibitor-resistant (PIR) clones from the human CAPAN1 pancreatic cancer cell line, which carries the protein-truncating c.6174delT frameshift mutation. PIR clones could form DNA-damage-induced RAD51 nuclear foci and were able to limit genotoxin-induced genomic instability, both hallmarks of a competent HR pathway. New BRCA2 isoforms were expressed in the resistant lines as a result of intragenic deletion of the c.6174delT mutation and restoration of the open reading frame (ORF). Reconstitution of BRCA2-deficient cells with these revertant BRCA2 alleles rescued PARP inhibitor sensitivity and HR deficiency. Most of the deletions in BRCA2 were associated with small tracts of homology, and possibly arose from error-prone repair caused by BRCA2 deficiency. Similar ORF-restoring mutations were present in carboplatin-resistant ovarian tumours from c.6174delT mutation carriers. These observations have implications for understanding drug resistance in BRCA mutation carriers as well as in defining functionally important domains within BRCA2.     

Abundance and diversity of microbial life in ocean crust

Oceanic lithosphere exposed at the sea floor undergoes seawater-rock alteration reactions involving the oxidation and hydration of glassy basalt. Basalt alteration reactions are theoretically capable of supplying sufficient energy for chemolithoautotrophic growth. Such reactions have been shown to generate microbial biomass in the laboratory, but field-based support for the existence of microbes that are supported by basalt alteration is lacking. Here, using quantitative polymerase chain reaction, in situ hybridization and microscopy, we demonstrate that prokaryotic cell abundances on seafloor-exposed basalts are 3-4 orders of magnitude greater than in overlying deep sea water. Phylogenetic analyses of basaltic lavas from the East Pacific Rise (9 degrees N) and around Hawaii reveal that the basalt-hosted biosphere harbours high bacterial community richness and that community membership is shared between these sites. We hypothesize that alteration reactions fuel chemolithoautotrophic microorganisms, which constitute a trophic base of the basalt habitat, with important implications for deep-sea carbon cycling and chemical exchange between basalt and sea water.     

Structure of a beta1-adrenergic G-protein-coupled receptor

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.