The Drosophila orthologue of progeroid human WRN exonuclease, DmWRNexo, cleaves replication substrates but is inhibited by uracil or abasic sites: Analysis of DmWRNexo activity in vitro

• Published in: AGE 2013-06, Vol.35 (3), p.793-806
• Main Authors: Mason, Penelope A., Boubriak, Ivan, Robbins, Timothy, Lasala, Ralph, Saunders, Robert, Cox, Lynne S.
• Format: Article
• Language: English
• Subjects: Aging; Animals; Biomedical and Life Sciences; Catalytic Domain - genetics; Cell Biology; Deoxyribonucleic acid; DNA; DNA repair; DNA Replication - genetics; DNA, Single-Stranded - genetics; Drosophila; Drosophila - genetics; Drosophila - metabolism; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Enzymes; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - metabolism; Exonucleases - genetics; Exonucleases - metabolism; Gene Expression Regulation; Genotype & phenotype; Geriatrics/Gerontology; Humans; Insects; Life Sciences; Medical disorders; Medical research; Metabolism; Molecular Medicine; Mutation; Phenotype; Proteins; RecQ Helicases - genetics; RecQ Helicases - metabolism; Sequence Analysis, DNA; Statistical analysis; Studies; Uracil - pharmacology; Werner Syndrome - drug therapy; Werner Syndrome - genetics; Werner Syndrome - metabolism; Werner Syndrome Helicase
• ISSN: 0161-9152; 2509-2715; 1574-4647; 2509-2723
• DOI: 10.1007/s11357-012-9411-0

Werner syndrome (WS) is a rare late-onset premature ageing disease showing many of the phenotypes associated with normal ageing, and provides one of the best models for investigating cellular pathways that lead to normal ageing. WS is caused by mutation of WRN , which encodes a multifunctional DNA replication and repair helicase/exonuclease. To investigate the role of WRN protein’s unique exonuclease domain, we have recently identified DmWRNexo, the fly orthologue of the exonuclease domain of human WRN. Here, we fully characterise DmWRNexo exonuclease activity in vitro , confirming 3′–5′ polarity, demonstrating a requirement for Mg 2+ , inhibition by ATP, and an ability to degrade both single-stranded DNA and duplex DNA substrates with 3′ or 5′ overhangs, or bubble structures, but with no activity on blunt ended DNA duplexes. We report a novel active site mutation that ablates enzyme activity. Lesional substrates containing uracil are partially cleaved by DmWRNexo, but the enzyme pauses on such substrates and is inhibited by abasic sites. These strong biochemical similarities to human WRN suggest that Drosophila can provide a valuable experimental system for analysing the importance of WRN exonuclease in cell and organismal ageing.