- 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 -www.sdu.dk/darc
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 -www.sdu.dk/darc
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 -www.sdu.dk/darc
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 -www.sdu.dk/darc
Method outline
γ-H2AX DAPI
6,3 Gy Gamma-irradiation
Unirradiated control
Unirradiated
DSB Response
γH2AX
Irradiated
Twin study:DNA repair (Comet assay)DNA damage responseMitochondrial function IR
DNA RepairTMRM
+
-
- 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 -www.sdu.dk/darc
Mitochondrial membrane potential CTRL cells
No of cells/well
Mem
bran
e po
tenti
al
- 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 -www.sdu.dk/darc
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 -www.sdu.dk/darc
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 -www.sdu.dk/darc
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 -www.sdu.dk/darc
Effect of sumoylation on WRN helicase and strand-annealing activity
SUMO-1SUMO-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 -www.sdu.dk/darc
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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