Abstract
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.
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Abbreviations
- BSA:
-
Bovine serum albumin
- DTT:
-
Dithiothreitol
- ECL:
-
Enhanced chemiluminescence
- HRP:
-
Horse radish peroxidase
- IPTG:
-
Isopropyl β-d-1-thiogalactopyranoside
- PBS:
-
Phosphate buffered saline
- TILLING:
-
Targeting Induced Local Lesions IN Genomes
- WS:
-
Werner syndrome
- WRN:
-
Protein mutated in Werner syndrome
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Acknowledgments
We thank Christine Borer for technical support, and Tony Willis for protein sequencing. This work was supported by the Biotechnology and Biological Sciences Research Council [grant numbers BB/E000924/1, BB/E002072/1 and BB/E016995/1].
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Boubriak, I., Mason, P.A., Clancy, D.J. et al. DmWRNexo is a 3′–5′ exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease. Biogerontology 10, 267–277 (2009). https://doi.org/10.1007/s10522-008-9181-3
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DOI: https://doi.org/10.1007/s10522-008-9181-3