Acknowledgements

I would like to thank xxxxxxxxxxxxxxxxxxxxxxxxxx

Name Student identity number: xxx

Contents

Abstract4

Preface5

  1. Introduction6
  2. RecQ helicases8
  3. The RecQ helicase family8
  4. Human RecQ helicases8
  5. WRN9
  6. Werner syndrome9
  7. The WRN protein11
  8. The functions of WRN in DNA repair14
  9. DNA repair pathways14
  10. BER and SSBR17
  11. NER19
  12. MMR21
  13. DSBR23
  14. ICL repair26
  15. Other functions of WRN33
  16. Replication33
  17. Transcription and chromatin structure34
  18. Telomere maintenance35
  19. Post-translational modificationof WRN36
  20. Phosphorylation of WRN36
  21. Acetylation of WRN37
  22. SUMOylation37
  23. Protein SUMOylation at a glance37
  24. The SUMO family of proteins38
  25. The SUMOylation pathway39
  26. Mechanisms of SUMO paralogue specificity44
  27. Consequences of SUMOylation46
  28. Interplay between SUMOylation and ubiquitination48
  29. SUMOylation and interplay with other post-translational modifications 50
  30. SUMOylation of WRN52
  31. Materials and Methods54
  32. Buffers and solutions54
  33. Cells lines and cell culture conditions54
  34. Plasmid constructs54
  35. Primers and PCR55
  36. Enzymes and chemicals56
  37. Antibodies56
  38. Protocols57
  39. Results61
  40. Project overview61
  41. Subcloning of WRN61
  42. Generation of a quadruple potential SUMOylation site WRN mutant63
  43. Overexpression of SUMO-2 produces high-molecular weight WRN species65
  44. WRN is SUMO-2/3 conjugated in vivo and SUMOylation of WRN is induced

by heat-shock67

9.6.Cisplatin exposure induces SUMO-2 modification of WRN70

9.7.Cells expressing SUMOylation-deficient WRN are impaired in the response

to replication-associated DNA double strand breaks.73

9.8.Cells expressing SUMOylation-deficient WRN are hypersensitive to cisplatin76

  1. Discussion and conclusion78
  2. Future perspectives85

Abbreviations89

References90

Supplementary102

S1. BLM and Bloom Syndrome102

S2. RECQ4 and Rothmund-Thomson Syndrome 104

S3. RECQ1 106

S4. RECQ5 106

S5. BLM post-translational modifications 107

S6. SUMOylation of BLM 108

Abstract

Werner Syndrome (WS) is a rare autosomal recessive disease characterized by ……………….

Preface

Mutations in the genes encoding proteins involved in the maintenance of genomic integrity are the cause of several progeroid syndromes that

This Master thesis can be seen as made up of two parts. The first part reviews the current understanding of the …………. The second part covers the experimental investigations that …..

Needless to say……

1. Introduction

As the process of senescence is complex, and may exist for a variety of different reasons, it can possibly derive from

2. RecQ helicases

2.1. The RecQ helicase family

The helicase class of enzymes are ………………………..

2.2. Human RecQ helicases

Human cells possess five distinct …………………………

3. WRN

3.1. Werner Syndrome

WS is a rare autosomal recessive ………..

3.2. The WRN protein

WRN is a 1432 amino acid protein with a …………………

Oligomeric states of WRN

The quaternary structure normally adopted by WRN ……….

Cellular localization of WRN

WRN is a nuclear protein but the subnuclear localization …….

4. The functions of WRN in DNA repair

4.1. DNA repair pathways

DNA in cells is continuously exposed to ………..

4.2. BER and SSBR

The BER process consists of ………..

4.3. NER

So far there has been little evidence for ………

4.4. MMR

The MMR pathway that removes base-base mismatches can be …………..

4.5. DSBR

The common, but toxic and mutagenic DNA DSB lesions are …………

Non-homologous end-joining (NHEJ)

Both exonuclease and helicase ……..

4.6. ICL repair

ICLs are very cyto- and genotoxic DNA lesions formed by ……….

Cisplatin

Cisplatin (cis-platinum(II) diamminedichloride) ………..

Mitomycin C

MMC is another commonly used crosslinking agent …………

Psoralen

Psoralen requires exposure ………..

Model for mammalian ICL repair

ICL repair in mammals is not yet well ………..

WRN and ICL repair

There is considerable evidence ………

5. Other functions of WRN

5.1. Replication

During replication of the genome the DNA …………………………

5.2. Transcription and chromatin structure

5.3. Telomere maintenance

Telomeres are specialized nucleoprotein structures with ……….

6. Post-translational modification of WRN

6.1. Phosphorylation of WRN

Phosphorylation is one of the most common forms of ……….

6.2. Acetylation of WRN

Acetylation of WRN by the transcriptional co-activator p300 in response to ………..

.

7. SUMOylation

7.1. Protein SUMOylation at a glance

SUMOylation is a reversible post-translational modification that …..

7.2. The SUMO family of proteins

SUMO (Small Ubiquitin-related MOdifier) proteins are unusual ……..

7.3. The SUMOylation pathway

The SUMOylation process results in ..

SUMOylation (SUMO conjugation)

Before the newly synthesized pro-SUMO proteins can …..

DeSUMOylation (SUMO deconjugation)

As described, the SUMO pathway is …

7.4. Mechanisms of SUMO paralogue specificity

Although some proteins are preferentially conjugated to one SUMO paralogue, many ………..

7.5. Consequences of SUMOylation

The functional effect and consequence of SUMO modification on a …..

SUMO-interacting motif (SIM)

As mentioned, SUMO itself may contribute to …….

7.6. Interplay between SUMOylation and ubiquitination

While the post-translational ubiquitin modification performs ………

Indirect co-regulation

An antagonistic relationship is one possible mode of ……….

Direct co-regulation

SUMOylation and ubiquitination can directly affect …………

.

7.7. SUMOylation and interplay with other post-translational modifications

Acetylation and SUMO

Acetylation of lysine residues can regulate ……………….

Phosphorylation and SUMO

Phosphorylation of proteins is now understood to …….

7.8. SUMOylation of WRN

The first report that WRN was a target for …………

8. Materials and Methods

8.1. Buffers and solutions

BSA-PBST: PBST + 5 % w/v BSA

HBS (2X): 274 mM NaCl, 10 mM KCl, 1.4 mM Na2HPO4, 12 mM glucose, 42 mM HEPES, adjust to pH 7.05

IP wash buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Igepal CA-630 ……………………..

8.2. Cells lines and cell culture conditions

E. coli cells

Prior to use all XL1-Blue and XL10-GoldE. coli cells …..

Human cells

Prior to use, all human HEK293T ….

8.3. Plasmid constructs

pBSCA-6XHis WRN expression vector (Figure 16) was generously provided …..

8.4. Primers and PCR

PCR

A ~500 bp sequence of WRN cDNA was ….

.

Touchdown PCR

Full-length WRN cDNA with …..

PCR Site-directed mutagenesis

PCR site-directed mutagenesis amplification was performed on .

DNA sequencing

The fluorescence-based cycle sequencing reactions for use in the DNA sequencing were made with ………

8.5. Enzymes and chemicals

Enzymes

Dpn I restriction enzyme …..

Chemicals

Cisplatin (cis-Platinum(II) diamminedichloride, Sigma) …..

8.6. Antibodies

The primary antibodies used to detect WRN protein were ………

8.7. Protocols

Note that while …

Agarose gel electrophoresis

1% agarose gels were cast with ………….

Transformation of E. coli

Heat-shock competent XL1-Blue E. coli or …………

Harvesting of plasmids from E. coli

Bacterial colonies were cultured in …………..

Transient transfections

Lipofectamine 2000-based transfection

Culture dishes were seeded with ………

Calcium Phosphate-based transfection

Culture dishes were ………….

.

Preparation of whole cell extracts

………………….

9. Results

9.1. Project overview

As previously described, WRN is involved in multiple ………

9.2. Subcloning of WRN

Numerous attempts were made to introduce ……..

9.3. Generation of a quadruple potential SUMOylation site WRN mutant

WRN has 117 lysine residues and ……….

9.4. Overexpression of SUMO-2 produces high-molecular weight WRN species

It has previously been shown that……..

9.5. WRN is SUMO-2/3 conjugated in vivo and SUMOylation of WRN is induced by heat-shock

One way to determine if the ………

9.6. Cisplatin exposure induces SUMO-2 modification of WRN

WRN has been extensively studied and characterized ….

9.7. Cells expressing SUMOylation-deficient WRN are impaired in the response to replication-associated DNA double-strand breaks.

WS cells are known to be ……

9.8. Cells expressing SUMOylation-deficient WRN are hypersensitive to cisplatin

If SUMOylation of WRN is important for .

Cisplatin

HEK293T cells were …..

Camptothecin

HEK293T cells were …….

10. Discussion and conclusion

WRN SUMOylation

Given that WRN is known to be …..

Possible roles of WRN and WRN SUMOylation in ICL repair

WS cells are hypersensitive to ….

.

Interstrand crosslinks in the premature aging phenotype of Werner Syndrome patients

The hallmark of Werner Syndrome is premature aging…….

SUMOylated WRN in the response to replication-dependent DSBs

WS cells are hypersensitive to CPT………

Conclusion

In conclusion, WRN can be found ………

11. Future perspectives

Aspects of WRN SUMOylation

Now that there is compelling evidence that WRN ……

Stably transfected isogenic cell lines

One very valuable approach would be the ….

WRN at telomeres

While there is little data on the effect of ……

Final words

The study of SUMO conjugated proteins is challenging and complex. The issue of ………

Abbreviations

1

4-NQO, 4-nitroquinoline-1-oxide

8-oxoA, 8-oxoadenine

8-oxoG, 8-oxoguanine

ALT, alternative lengthening of telomeres

………………………

………………………

………………………

………………………

………………………

………………………

………………………

………………………

………………………

………………………

………………………

………………………

WS, Werner syndrome

1

References

1

Ababou, M., Dutertre, S., Lecluse, Y., Onclercq, R., Chatton, B., Amor-Gueret, M., 2000.ATM-dependent phosphorylation and accumulation of endogenous BLM protein in response to ionizing radiation.Oncogene.19, 5955-63.

Agbor, T.A., Taylor, C.T., 2008. SUMO, hypoxia and the regulation of metabolism.Biochem Soc Trans. 36, 445-8.

Ahn, B., Harrigan, J.A., Indig, F.E., Wilson, D.M., 3rd, Bohr, V.A., 2004. Regulation of WRN helicase activity in human base excision repair.J Biol Chem. 279, 53465-74.

Ammazzalorso, F., Pirzio, L.M., Bignami, M., Franchitto, A., Pichierri, P., ATR and ATM differently regulate WRN to prevent DSBs at stalled replication forks and promote replication fork recovery. EMBO J.

Supplementary

Contents

S1. BLM and Bloom Syndrome

S2. RECQ4 and Rothmund-Thomson Syndrome

S3. RECQ1

S4. RECQ5

S5. BLM post-translational modifications

S6. SUMOylation of BLM

S1. BLM and Bloom Syndrome

BS is a very rare autosomal recessive disorder with ……..

S2. RECQ4 and Rothmund-Thomson Syndrome

RTS is a rare autosomal recessive disorder characterized by …….

S3. RECQ1

No genetic disorders have so far been associated with …….

S4. RECQ5

RECQ5β is the largest of the three (α, β and γ) …….

S5. BLM post-translational modifications

BLM is phosphorylated and accumulates in an……..

S6. SUMOylation of BLM

A 2005 study identified BLM as a target for ……

1