Molecular basis of replicative senescence
Co-workers: Dr Penelope Mason, Mr Ralph Lasala, Ms Hayley Lees
Replicative senescence is loss of ability of cells to copy their DNA, that underlies and is thought to cause human ageing. We study progeroid Werner’s syndrome (WS), where cell senescence and early onset of many age-related symptoms in the patients result from loss of a single protein, the WRN helicase/exonuclease that is involved in DNA replication, recombination and repair.In collaboration with Dr Saunders (Open University), we have identified a novel WRN exonuclease in Drosophila and shown that flies homozygous for a strongly hypomorphic allele are hyperrecombinant and hypersensitive to the replication fork collapsing agent, camptothecin.
An important role for DmWRNexo in early development is suggested by maternal effect lethality during embryogenesis. We demonstrate for the first time the importance of the exonuclease activity of WRN in maintaining genome stability.We have developed a novel fluorescence-based exonuclease assay, and demonstrated that DmWRNexo is a 3’-5’ exonuclease that acts on replication–intermediate type DNA substrates, Mutation of putative active site residues abolishes nuclease activity, while mutation of a surface residue (D229V) alters the enzyme’s specificity. We are now identifying protein partners of DmWRNexo.
- Boubriak I, Mason PA, Clancy DJ, Dockray J, Saunders RD, Cox LS. DmWRNexo is a 3'-5' exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease. Biogerontology. 2009 Jun;10(3):267-77
- Saunders RD, Boubriak I, Clancy DJ, Cox LS. (2008) Identification and characterization of a Drosophila ortholog of WRN exonuclease that is required to maintain genome integrity. Aging Cell. 2008 Jun;7(3):418-25. Epub 2008 Mar 11. PMID: 18346216
- Cox LS, Clancy DJ, Boubriak I, Saunders RD. (2007) Modeling Werner Syndrome in Drosophila melanogaster: hyper-recombination in flies lacking WRN-like exonuclease. Ann N Y Acad Sci. 2007 Nov;1119:274-88. PMID: 18056975
- Cox LS, Faragher RG. (2007) From old organisms to new molecules: integrative biology and therapeutic targets in accelerated human ageing. Cell Mol Life Sci. 2007 Oct;64(19-20):2620-41. Review. PMID: 17660942
- Rodriguez-Lopez AM, Whitby MC, Borer CM, Bachler MA, Cox LS. (2007) Correction of proliferation and drug sensitivity defects in the progeroid Werner's Syndrome by Holliday junction resolution. Rejuvenation Res. 2007 Mar;10(1):27-40. PMID: 17378750
Figure 1: (A) Replicating DNA molecules from a patient with Werner's syndrome show defects in the movement of replication forks; DNA was "combed" onto glass slides then stained for a label present in newly replicated DNA. Images were taken by confocal microscopy. (B) Such defects in replication lead to fork stalling and collapse into 4-way DNA Holliday junctions. (C) Rapid resolution of Holliday junctions by a bacterial enzyme, RusA, restores DNA replication capacity to Werner's syndrome cells; a mutant form of RusA (D70N) has no effect.
Figure 2: WRN co-localizes with an essential replication factor, PCNA, at replication foci in normal human fibroblasts. These sites represent replication factories as shown by immuno-electron microscopy (EM)
Figure 3: High levels of DNA recombination in a fly lacking functional Drosophila WRN exonuclease are detected by counting cells expressing the "multiple wing hairs" marker in the adult wing (region outlined in red)
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