Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - DmWRNexo is a 3'aEuro"5' exonuclease
T2 - phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease
AU - Boubriak, Ivan
AU - Mason, Penelope A.
AU - Clancy, David J.
AU - Dockray, Joel
AU - Saunders, Robert D. C.
AU - Cox, Lynne S.
N1 - Jun DmWRNexo is a 3'aEuro"5' exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease Boubriak, Ivan Mason, Penelope A. Clancy, David J. Dockray, Joel Saunders, Robert D. C. Cox, Lynne S. International Workshop on Cell Semescemce - Future of Ageing JUL 07-08, 2008 Oxford, ENGLAND Sp. Iss. SI
PY - 2009/6
Y1 - 2009/6
N2 - 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'aEuro"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.
AB - 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'aEuro"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.
KW - Werner syndrome
KW - WRN
KW - Exonuclease
KW - CG7670
KW - Aging
KW - Ageing
KW - Recombination
KW - Drosophila
KW - DmWRNexo
KW - REPLICATION FORK PROGRESSION
KW - WERNER-SYNDROME PROTEIN
KW - SYNDROME GENE
KW - FUNCTIONAL GENOMICS
KW - RECQ HELICASE
KW - SWISS-MODEL
KW - DNA-DAMAGE
KW - FIBROBLASTS
U2 - 10.1007/s10522-008-9181-3
DO - 10.1007/s10522-008-9181-3
M3 - Journal article
VL - 10
SP - 267
EP - 277
JO - Biogerontology
JF - Biogerontology
SN - 1389-5729
IS - 3
ER -