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DmWRNexo is a 3'aEuro"5' exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease

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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. et al.
In: Biogerontology, Vol. 10, No. 3, 06.2009, p. 267-277.

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Boubriak I, Mason PA, Clancy DJ, Dockray J, Saunders RDC, Cox LS. DmWRNexo is a 3'aEuro"5' exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease. Biogerontology. 2009 Jun;10(3):267-277. doi: 10.1007/s10522-008-9181-3

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@article{90e1d9f7228e41aab5f1ecbce13694e7,
title = "DmWRNexo is a 3'aEuro{"}5' exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease",
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'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.",
keywords = "Werner syndrome, WRN, Exonuclease, CG7670, Aging, Ageing, Recombination, Drosophila, DmWRNexo, REPLICATION FORK PROGRESSION, WERNER-SYNDROME PROTEIN, SYNDROME GENE, FUNCTIONAL GENOMICS, RECQ HELICASE, SWISS-MODEL, DNA-DAMAGE, FIBROBLASTS",
author = "Ivan Boubriak and Mason, {Penelope A.} and Clancy, {David J.} and Joel Dockray and Saunders, {Robert D. C.} and Cox, {Lynne S.}",
note = "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",
year = "2009",
month = jun,
doi = "10.1007/s10522-008-9181-3",
language = "English",
volume = "10",
pages = "267--277",
journal = "Biogerontology",
issn = "1389-5729",
publisher = "Springer Netherlands",
number = "3",

}

RIS

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 -