Genetic Syndrome

David M. Wilson III, Ph.D. Laboratory of Molecular Gerontology National Institute on Aging Baltimore, Maryland

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Sources of DNA Damage

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA CONSEQUENCES OF DNA DAMAGE

STRESS Exogenous Endogenous

DNA Lesion TRANSCRIPTION: Blockage or Error

UNREPAIRED REPLICATION: Blockage or Error

Genetic Instability Cellular Dysfunction Cell Death

CANCER

NEURODEGENERATION

AGING

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA DNA DAMAGE AND REPAIR IN THE NUCLEUS

Reactive Oxygen Species UV Light Ionizing Radiation Alkylating Agents Carcinogens Crosslinkers Spontaneous Decay MM Gox U

AP Site BASE EXCISION NUCLEOTIDE MISMATCH RECOMBINATION REPLICATION REPAIR EXCISION REPAIR REPAIR FORK ARREST REPAIR & CELL CYCLE Short- Long- SSBR Homologous NHEJ CHECKPOINTS patch patch - Global Specific DIRECT REVERSAL (O6-methylguanine TCR methyltransferase; photolyase)

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA MAMMALIAN DNA REPAIR Nuclear vs Mitochondrial

PATHWAY SUBPATHWAY Nuclear Mito

Long-patch Y Y Base excision repair Short-patch Y Y General genome Y N Nucleotide excision repair Gene-specific Y N Transcription associated Y N

Mismatch repair Y y

Homologous recombination Y ? Recombination repair Non-homologous end-joining Y ?

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA PREMATURE AGING DISORDERS

Werner Syndrome

Cockayne Syndrome

Rothmund Hutchinson- Thomson Gilford Syndrome Progeria

All stem from defects in DNA presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA XP/CS damage responses. Underlying Hypothesis:

The clinical symptoms associated with the premature aging syndromes arise from inefficient global and/or region-specific repair of endogenous DNA damage.

Vilhelm A. Bohr, MD, PhD

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Age-Associated Diseases

• • • Arthritis • • Osteoporosis • Type 2 diabetes • Hypertension • Alzheimer disease (dementia)

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA (CS)

• Rare autosomal recessive . – two strict complementation groups, A and B. • Segmental premature aging disease. – life expectancy of ~12.5 years. • Major clinical features: – impaired growth/development (cachectic dwarfism) – multisystem progressive degeneration (neurological abnormalities) – no increased cancer incidence

• Molecular features: http://www.cockaynesyndrome.net/ – sensitivity to sunlight – failure to recover RNA synthesis after UV light exposure

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Three Prevailing Theories for CS

Theory 1. CS facilitate the repair of transcription-blocking lesions in a process called transcription-coupled DNA repair.

Theory 2. CS proteins operate as direct regulators of transcription, in part through an association with RNA polymerases.

Theory 3. CS proteins promote the efficient repair of endogenous (oxidative) DNA damage, both in the nuclear and mitochondrial compartments.

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Cockayne Syndrome B (CSB)

1 I IA II III IV V VI 1493

Acidic region (aa 356-394) Gly rich region (aa 442-450) Two putative NLS (aa 466-481 + aa 1038-1055) The 7 ATPase domains (aa 529-949)

• SWI2/SNF2 family member with conserved -like ATPase motifs. • No helicase activity, but proposed role in remodeling. • Homologous DNA strand pairing activity. • Interacts with (not limited to) RNA Pol II, XP-A, XP-G, TFIIE, TFIIH and several splicing factors. • Participates in transcription-coupled and global genome DNA repair, and in general transcription.

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA CSB Functions in Nuclear Base Excision Repair (BER)

STEPS CORE PARTICIPANTS 5’

1. Base Excision Glycosylase OGG1 NEIL1

2. AP Site Incision dRP APE1

3. Gap-filling POL CSB

4. dRP Removal POL PARP1

Dianov et al., Nucleic Acids Res, 1999 5. Nick Ligation LIG3/XRCC1 Tuo et al., J. Biol. Chem., 2001 Thorslund et al., Mol. Cell. Biol., 2005 Wong et al., Nucleic Acids Res, 2007 Muftuoglu et al., J. Biol. Chem., 2009

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA CSB Interacts and Exists in a Common Protein Complex with APE1

0.6 0.7 0.8 0.9 1.0 ng

Ape1

c-Abl ECFP   APE1 IP : IP : IP Control Input Control Input FEN1 IB: CSB IB: Ape1 p53

PCNA IB: Ape1 IB: CSB CSB 1 2 3 1 2 3

Negative interactors: p53, PCNA, Ku70/80, WRN, NBS1, TRF1, Rad51, and RPA.

Wong et al., Nucleic Acids Research, 1997

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA CSB Stimulates APE1 AP Site Incision Activity

11 42 bubble comp APE1 - + - + + + + CSB - - + S

P

120

100

80

60

Percent Incision Percent 40

20

0 APE1 CSB APE1- APE1- APE1- APE1- CSB CSB CSB CSB ATP-independent 3:1 1:1 3:10 1:10

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA CSB Mutant Cells Exhibit Increased Sensitivity to BER DNA- Damaging Agents: MMS and HmdU

1000 CSB-WT 1000 CSB-WT CSB-V CSB-V

100 100

10 10 Percent Survival Percent Percent Survival Percent

1 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 5 10 15 20 MMS concentration (mM) HmdU concentration (mM)

CSB mutant cell line = CS1AN.S3.G2, an SV40-transformed human skin fibroblast line MMS = methylmethane sulfonate HmdU = hydroxymethyl deoxyuridine

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Evidence for Role of CS Proteins in Oxidative Stress Resistance & BER

• CS cells are mildly hypersensitive to ionizing radiation, hydrogen peroxide, and potassium bromate (oxidizing agents).

• CS cells exhibit inefficient repair of oxidative DNA base damage, such as 8-oxoguanine, 8-oxoadenine and cyclodeoxypurines.

• The hypersensitivity to MMS and HmdU indicates a role for CSB in the efficient processing of BER DNA intermediates (likely AP sites and/or SSBs).

• CSB functionally interacts with NEIL1, APE1, and PARP1, thereby modulating BER efficiency.

• Data indicates a role for the CSB-APE1 interaction in gene-specific repair (possibly TCR) of AP sites, not likely in global repair.

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA MITOCHONDRIAL THEORY OF AGING mtDNA damage/

Mitochondrial dysfunction 8-oxodG in rat liver mtDNA Elevated ROS production 25

20 dG

5 Cellular dysfunction 15

10 8-oxodG/10 Aging 5

Age (months) 6 12 23

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Co-localization of CSB with TFAM in Mitochondria After Menadione Treatment of HeLa Cells

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USAAamann et al., FASEB J, 2010 Decreased Repair Efficiency in the Membrane-bound Fraction of CSB-deficient Mitochondria

Similar results with uracil and 5-hydroxy-uracil base lesions.

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Increased Mitochondrial Mutagenesis in CSB Knockdown Cells

CAP = chloramphenicol

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Roles of CSB in Maintenance of Cellular Homeostasis

• Transcription • Transcription-coupled repair • Base excision repair

• Transcription • Base excision repair •

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Age-Associated Diseases

• Cardiovascular disease • Cancer • Arthritis • Cataracts HIV-Related • Osteoporosis Premature Aging • Type 2 diabetes Phenotypes • Hypertension Alzheimer disease (dementia) • Likely stem from antiretroviral toxicities (mitochondrial dysfunction), direct effects of the virus invasion, and chronic inflammatory state

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA Acknowledgments

Heng Kuan-Wong, PhD Vilhelm Bohr & Many LMG Colleagues

Maria D. Aamann, PhD

Morten Scheibye-Knudsen, MD

presented at the 2nd Int Workshop on HIV & Aging, 27 - 28 Oct 2011, Baltimore, USA