- 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.

- 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

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 -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

+

-

DNA repair activity & DNA damage responseSSB DSB

gH2AXXRCC1

- 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