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Investigating DNA Repair and Aging through Twin Studies: Insights into WRN Helicase Regulation

This project explores the variability in human aging through a twin study focused on DNA repair efficiency and the posttranslational regulation of the WRN helicase. By analyzing blood samples from monozygotic and dizygotic twins, we assess DNA damage responses, mitochondrial function, and how factors such as sumoylation affect WRN's role in DNA repair. Our comprehensive approach utilizes comet assays, flow cytometry, and immunocytochemistry to uncover the mechanisms influencing aging and cellular resilience in response to DNA damage.

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Investigating DNA Repair and Aging through Twin Studies: Insights into WRN Helicase Regulation

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  1. I. Twin study on DNA repair efficiency and healthy human aging II. Posttranslational regulation of the RecQhelicase WRN Christian H. Garm, MSc, Ph.d. student Enrolled: March 2009 Supervisors: Kaare Christensen/SDU Tinna Stevnsner/AU VELUX FONDEN Why do we age so differently? Funded by:

  2. Project outline • Twin study • DNA repair efficiency • DNA damage response • Mitochondrial function • Posttranslational regulation of the RecQhelicase WRN • Regulation of WRN functions • Protein interaction • Intercellular localization

  3. DNA repair mechanisms

  4. The twin study DNA repair efficiency • Comet Assays (neutral & alkaline) DNA damage response • Flow Cytometry & Immunocytochemistry γH2AX, 53BP1, XRCC1 • Mitochondrial function • Mitochondrial membrane potential • Bloodsamples from MADT twins are currently collected in Aarhus • Peripheral Blood Mononuclear Cells • Lymphocytes • MZ; DZ; Old; Young

  5. Irradiated Method outline • Twin study: • DNA repair (Comet assay) • DNA damage response • Mitochondrial function DSB Response γH2AX IR Unirradiated γ-H2AX DAPI DNA Repair + Unirradiated control TMRM 6,3 Gy Gamma-irradiation -

  6. DNA repair activity & DNA damage response DSB SSB XRCC1 gH2AX

  7. Mitochondrial membrane potential CTRL cells Membrane potential No of cells/well

  8. II. Posttranslational regulation of the RecQhelicase WRN.

  9. WRN protein and DNA repair • Diverse roles of WRN in DNA repair • DSB repair • SSB repair • Replication forks • Telomere repair • WRN is a modular multifunctional enzyme • DNA dependent ATPase • 3´-5´ helicase • 3´-5´ exonuclease • Strand annealing • Strand exchange

  10. Posttranslational modification of the WRN protein • Sumoylation • Covalent conjugation of SUMO proteins at lysine residues • SUMO-1 • SUMO-2/3: 96 % identity • SUMO-4 • Functions: • Cellular localization • Protein interactions • Catalytic activity • Proteinstability • Conclusion: • Mono-conjugation of SUMO-1 • Poly-conjugation of SUMO-2/3 chains

  11. Effect of sumoylation on WRN helicase and strand-annealing activity SUMO-2/3 Helicase SUMO-1 SUMO-2/3 Strand-annealing Conclusions: • WRN helicase activity is stimulated by conjugation of SUMO-2/3 but not SUMO-1 • SUMO-2/3 conjugation reduces the DNA strand-annealing activity of WRN Work performed by Rikke Frölich

  12. Ongoing and future studies • Twin project • Intertwin differences in DNA repair & mitochondrial functions (MZ) • Correlate w. information from interviews. • Environmental contribution to aging. • Intertwin differences in MZ vs. DZ twins • Genetic contribution to aging • Differences between young and old twins • Regulation with age • WRN project • Regulation of WRN functions & interactions by sumoylation • Characterization of sumoylation deficient WRN • Protein interactions • Cellular localization • Telomere function

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