- D A N I S H A G I N G R E S E A R C H C E N T E R - I. Twin study on DNA repair efficiency and healthy human aging II. Posttranslational regulation of the RecQ helicase WRN Why do we age so differently? Christian H. Garm, MSc, Ph.d. student Enrolled: March 2009 Supervisors: Kaare Christensen/SDU Tinna Stevnsner/AU VELUX FONDEN Funded by:

- D A N I S H A G I N G R E S E A R C H C E N T E R -  Twin study  DNA repair efficiency  DNA damage response  Mitochondrial function  Posttranslational regulation of the RecQ helicase WRN  Regulation of WRN functions  Protein interaction  Intercellular localization Project outline

- D A N I S H A G I N G R E S E A R C H C E N T E R - DNA repair mechanisms

- D A N I S H A G I N G R E S E A R C H C E N T E R - 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

- D A N I S H A G I N G R E S E A R C H C E N T E R - Method outline γ-H2AXDAPI 6,3 Gy Gamma- irradiation Unirradiated control Unirradiated DSB Response γH2AX Irradiated  Twin study:  DNA repair (Comet assay)  DNA damage response  Mitochondrial function IR DNA Repair TMRM + -

DNA repair activity & DNA damage response SSB DSB gH2AX XRCC1

- D A N I S H A G I N G R E S E A R C H C E N T E R - Mitochondrial membrane potential CTRL cells No of cells/well Membrane potential

- D A N I S H A G I N G R E S E A R C H C E N T E R - II. Posttranslational regulation of the RecQ helicase WRN.

- D A N I S H A G I N G R E S E A R C H C E N T E R - WRN protein and DNA repair  WRN is a modular multifunctional enzyme  DNA dependent ATPase  3´-5´ helicase  3´-5´ exonuclease  Strand annealing  Strand exchange  Diverse roles of WRN in DNA repair  DSB repair  SSB repair  Replication forks  Telomere repair

- D A N I S H A G I N G R E S E A R C H C E N T E R -  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  Protein stability  Conclusion:  Mono-conjugation of SUMO-1  Poly-conjugation of SUMO-2/3 chains Posttranslational modification of the WRN protein

- D A N I S H A G I N G R E S E A R C H C E N T E R - Effect of sumoylation on WRN helicase and strand-annealing activity SUMO-1 SUMO-2/3 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 SUMO-2/3Helicase Strand-annealing Work performed by Rikke Frölich

- D A N I S H A G I N G R E S E A R C H C E N T E R - 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