SummaryThe premature human aging Werner syndrome (WS) is caused by mutation of the RecQ-family WRN helicase, which is unique in possessing also 3′ ′ ′ ′ -5′ ′ ′ ′ exonuclease activity. WS patients show significant genomic instability with elevated cancer incidence. WRN is implicated in restraining illegitimate recombination, especially during DNA replication. Here we identify a Drosophila ortholog of the WRN exonuclease encoded by the CG7670 locus. The predicted DmWRNexo protein shows conservation of structural motifs and key catalytic residues with human WRN exonuclease, but entirely lacks a helicase domain. Insertion of a piggyBac element into the 5′ ′ ′ ′ UTR of CG7670 severely reduces gene expression. DmWRNexo mutant flies homozygous for this insertional allele of CG7670 are thus severely hypomorphic; although adults show no gross morphological abnormalities, females are sterile. Like human WS cells, we show that the DmWRNexo mutant flies are hypersensitive to the topoisomerase I inhibitor camptothecin. Furthermore, these mutant flies show highly elevated rates of mitotic DNA recombination resulting from excessive reciprocal exchange. This study identifies a novel WRN ortholog in flies and demonstrates an important role for WRN exonuclease in maintaining genome stability.

The ability of seeds to develop a mucilaginous layer when wetted by night-time dew, and to repair their DNA under these conditions, appear to be mechanisms that help maintain seed viability under harsh desert conditions.

It is proposed that desiccation tolerance in the embryo of seeds depends upon the capacity to repair damage to genomic DNA when the desiccated embryo is rehydrated. From a study of imbibed and hydrated embryos of rye (Secale cereale) and wild oat (Avena fatua) evidence is provided that it is neither the extent of water uptake by the cells, the ensuing stability of the DNA to desiccation, nor the onset of S-phase DNA synthesis in the first cell cycle of germination that determines whether the desiccated embryo will survive. It is shown that when α- and β-polymerases of DNA repair are inhibited by aphidicolin and dideoxythymidine-5'-triphosphate, respectively, a γ-irradiation-induced DNA fragmentation cannot be fully repaired. It is shown that in hydrated embryos, at a stage when desiccation tolerance is lost, embryo cells still repair irradiation-induced damage, but this repaired DNA is unstable to desiccation and cannot be rerepaired when water is again made available. The failure to re-repair on rehydration appears to be critical to embryo survival and successful germination.

The premature human ageing Werner's syndrome is caused by loss or mutation of the WRN helicase/exonuclease. We have recently identified the orthologue of the WRN exonuclease in flies, DmWRNexo, encoded by the CG7670 locus, and showed very high levels of mitotic recombination in a hypomorphic PiggyBac insertional mutant. Here, we report a novel allele of CG7670, with a point mutation resulting in the change of the conserved aspartate (229) to valine. Flies bearing this mutation show levels of mitotic recombination 20-fold higher than wild type. Molecular modelling suggests that D229 lies towards the outside of the molecule distant from the nuclease active site. We have produced recombinant protein of the D229V mutant, assayed its nuclease activity in vitro, and compared activity with that of wild type DmWRNexo and a D162A E164A double active site mutant we have created. We show for the first time that DmWRNexo has 3'-5' exonuclease activity and that mutation within the presumptive active site disrupts exonuclease activity. Furthermore, we show that the D229V mutant has very limited exonuclease activity in vitro. Using Drosophila, we can therefore analyse WRN exonuclease from enzyme activity in vitro through to fly phenotype, and show that loss of exonuclease activity contributes to genome instability.

We describe two unrelated patients with pyruvate dehydrogenase (PDH) deficiency attributable to mutations in the gene encoding the E1beta subunit of the complex. This is a previously unrecognised form of PDH deficiency, which most commonly results from mutations in the X-linked gene for the E1alpha subunit. Both patients had reduced immunoreactive E1beta protein and both had missense mutations in the E1beta gene. Activity of the PDH complex was restored in cultured fibroblasts from both patients by transfection and expression of the normal E1beta coding sequence.