Acknowledgements
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1
Contents
Abstract
4
Preface
5
1. Introduction
6
2. RecQ helicases
8
2.1. The RecQ helicase family
8
2.2. Human RecQ helicases
8
3. WRN
3.1. Werner syndrome
9
3.2. The WRN protein
11
4. The functions of WRN in DNA repair
14
4.1. DNA repair pathways
14
4.2. BER and SSBR
17
4.3. NER
19
4.4. MMR
21
4.5. DSBR
23
4.6. ICL repair
26
5. Other functions of WRN
33
5.1. Replication
33
5.2. Transcription and chromatin structure
34
5.3. Telomere maintenance
35
6. Post-translational modification of WRN
36
6.1. Phosphorylation of WRN
36
6.2. Acetylation of WRN
37
7. SUMOylation
37
7.1. Protein SUMOylation at a glance
37
7.2. The SUMO family of proteins
38
7.3. The SUMOylation pathway
39
7.4. Mechanisms of SUMO paralogue specificity
44
7.5. Consequences of SUMOylation
46
7.6. Interplay between SUMOylation and ubiquitination
48
7.7. SUMOylation and interplay with other post-translational modifications
50
7.8. SUMOylation of WRN
52
8. Materials and Methods
8.1. Buffers and solutions
2
9
54
54
8.2. Cells lines and cell culture conditions
54
8.3. Plasmid constructs
54
8.4. Primers and PCR
55
8.5. Enzymes and chemicals
56
8.6. Antibodies
56
8.7. Protocols
57
9. Results
61
9.1. Project overview
61
9.2. Subcloning of WRN
61
9.3. Generation of a quadruple potential SUMOylation site WRN mutant
63
9.4. Overexpression of SUMO-2 produces high-molecular weight WRN species
65
9.5. WRN is SUMO-2/3 conjugated in vivo and SUMOylation of WRN is induced
by heat-shock
9.6. Cisplatin exposure induces SUMO-2 modification of WRN
67
70
9.7. Cells expressing SUMOylation-deficient WRN are impaired in the response
to replication-associated DNA double strand breaks.
9.8. Cells expressing SUMOylation-deficient WRN are hypersensitive to cisplatin
73
76
10. Discussion and conclusion
78
11. Future perspectives
85
Abbreviations
89
References
90
Supplementary
102
S1. BLM and Bloom Syndrome
102
S2. RECQ4 and Rothmund-Thomson Syndrome
104
S3. RECQ1
106
S4. RECQ5
106
S5. BLM post-translational modifications
107
S6. SUMOylation of BLM
108
3
Abstract
Werner Syndrome (WS) is a rare autosomal recessive disease characterized by ……………….
4
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……
5
1. Introduction
As the process of senescence is complex, and may exist for a variety of different reasons, it can
possibly derive from
6
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 ………..
7
Figure 4 | DNA lesions and their repair by the four major DNA repair pathways in higher eukaryotes.
Cells have multiple DNA repair pathways that provide ……………
Figure 5 | Interaction of the RecQ helicases with DNA repair pathways.
Evidence indicates that the RecQ helicases interacts with all the major DNA damage processing and repair pathways,
including resolution of stalled replication forks.
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 ……….
8
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 ………..
.
9
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 ………
10
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-Gold E. coli cells …..
Human cells
Prior to use, all human HEK293T ….
8.3. Plasmid constructs
pBSCA-6XHis WRN expression vector (Figure 16) was generously provided …..
11
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 ………
12
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
13
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
14
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 ………
15
Abbreviations
4-NQO, 4-nitroquinoline-1-oxide
………………………
8-oxoA, 8-oxoadenine
………………………
8-oxoG, 8-oxoguanine
………………………
ALT, alternative lengthening of telomeres
………………………
………………………
………………………
………………………
………………………
………………………
………………………
………………………
………………………
16
WS, Werner syndrome
References
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.
17
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 ……
18