New insights in the difference between UVsensitive syndrome and Cockayne syndrome
through new-found functions for UVSSA
and USP7 in transcription coupled repair
Author:
Supervisor:
Examiner:
Master:
Vera Boersma, 3157113
Jurgen Marteijn, PhD, Erasmus MC
Puck Knipscheer, PhD, Hubrecht Institute
Cancer Genomics & Developmental Biology, UU
Cover picture adapted from environmental bio-detection products Inc.
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Cover picture adapted from environmental bio-detection products Inc.
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Abstract
DNA repair is an important mechanism in the cell to prevent mutations as a consequence of DNA
damage, and possible adverse effects due to these mutations. There are several diseases known to
be caused by defects in DNA repair pathways. Two of these diseases, UV sensitive syndrome and
Cockayne syndrome, are caused by defects in transcription coupled repair, a sub pathway of
nucleotide excision repair. Even though these diseases are caused by defects in the same pathway,
their clinical manifestations differ greatly. UVSS patients show mild sun sensitivity, where CS patients
show a more severe phenotype, including neurodegenerative symptoms. The causal distinction
between these two diseases has been a mystery for a long time. Recently a new gene has been
identified as the causal gene of UVSS, but not CS: UVSSA. Together with the DUB USP7, UVSSA
might have an important role in the ubiquitin-regulation of TCR factors such as CSB. Mutations in CSB
or CSA can cause Cockayne syndrome. It has been suggested that CSB and CSA might have
different functions next to their role in TCR. The discovery of UVSSA and the new role of USP7 in TCR
can shed a light on the distinction between UVSS and CS. Here I suggest that UVSSA only abolishes
the function of CSB in TCR, but leaves CSB intact to fulfill its other functions, where in CS, CSB is
dysfunctional in multiple processes, causing additional symptoms next to mild UV sensitivity.
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Table of Contents
Abstract ................................................................................................................................................... 3
Introduction............................................................................................................................................. 5
DNA repair ........................................................................................................................................... 5
Double strand break repair ............................................................................................................. 5
Single strand lesion repair ............................................................................................................... 6
DNA repair coupled to transcription ............................................................................................... 6
NER pathways .......................................................................................................................................... 7
Lesion recognition in GGR ................................................................................................................... 8
Lesion recognition in TCR .................................................................................................................... 8
CSB ................................................................................................................................................... 8
CSA................................................................................................................................................... 9
RNApolII regulation in TCR .............................................................................................................. 9
NER related diseases ......................................................................................................................... 10
UVSSA & USP7 in TC-NER .................................................................................................................. 11
Identification of UVSS-A in UVSSA................................................................................................. 11
Recruitment and interactions........................................................................................................ 11
UVSSA; a function in ubiquitin regulation of TC-NER .................................................................... 12
Proposing a new model for UVSSA and USP7 in TCR ............................................................................ 14
Implications on Cockayne syndrome vs. UV-sensitive syndrome ..................................................... 15
Concluding remarks ........................................................................................................................... 17
Acknowledgements ............................................................................................................................... 18
References ............................................................................................................................................. 19
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Introduction
DNA repair
DNA can be damaged by many
different intrinsic and extrinsic
causes, resulting in different
types of DNA damage (see
figure 1 for an overview).
Examples of extrinsic DNA
damaging factors are ionizing
radiation,
UV-light
and
genotoxic
chemicals
like
tobacco smoke (Hoeijmakers,
2001). UV-irradiation causes 64 photo products (6-4PP) and
cyclobutane pyrimidine dimers
(CPDs)
(Palomera-Sanchez,
2011). One of the intrinsic
causes of DNA damage are
reactive oxygen species (ROS),
Figure 1; Overview DNA damage: causes, types and repair (adapted
resulting in oxidized bases
from Hoeijmakers, 2001). Single-base lesions can be caused by X-rays,
(Hoeijmakers, 2001). When
ROS, alkylating agents and spontaneous reactions, they are repaired by
DNA damage is not restored
BER. Bulky adducts blocking transcription are caused by UV-light and
perfectly, mutations can occur,
polycyclic aromatic hydrocarbons and are repaired by NER. Double
possibly
leading
to
strand breaks are caused by X-rays or anti-tumor agents and are
chromosomal rearrangements.
repaired by HR or NHEJ. Replication errors cause mismatches,
Mutations and chromosomal
insertions and deletions which are repaired by mismatch repair.
rearrangements can even lead
to cancer. Persistent DNA damage can also lead to stalled RNA polymerases of replication forks. This
can result in cell cycle arrest or even apoptotic events, which could eventually lead to (accelerated)
aging. To prevent this, the cell is equipped with different specialized DNA repair pathways for each
type of damage (see figure 1 for an overview). These pathways differ in the triggering mechanism
specific to the lesion. Also the mechanism and thus efficiency of repair varies between the DNA
repair mechanisms (Hoeijmakers, 2001).
Double strand break repair
Double strand breaks (DSB) can be repaired by either non-homologous end joining (NHEJ) or
homologous recombination (HR). In NHEJ two ends of a DSB are ligated together by ligating enzymes
after processing of the broken DNA ends. This process is very error prone, since there is no check
whether the right chromosome ends are fused together. Sequence information might be lost due to
the deletion of nucleotides during the processing of the DNA ends (Lieber, 2010).
Homologous recombination (HR) is a process where the homologue sister chromatid is used as a
template to repair the damage. A strand of the broken chromosome invades the chromatid sister and
uses the complement strand as a template to transcribe the nucleotides to correctly repair the
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damage. This process is very accurate, but it can only be performed in G2/M when the chromosomes
have doubled prior to the damage and the chromatid sisters are thus still close together (Karpenshif,
2012).
Single strand lesion repair
Lesions only affecting one strand of the DNA are repaired by two different pathways: base excision
repair (BER) and nucleotide excision repair (NER).
BER repairs single bases damaged by alkylation, oxidization or spontaneous base loss. These
lesions mildly affect the DNA helix, and will cause no or little stalling of RNA polymerases. These
lesions are detected and excised by a DNA glycosylase. The resulting apyrmidinic and apurinic (AP)
sites need processing by DNA AP endonucleases and lyases to create 5’ and 3’ nicks at the excised
site. These sites are now compatible for further processing. A DNA polymerase will fill in the single
nucleotide gap, and finally DNA ligases will ligate the repaired part and the original strand together.
BER can be subdivided into two sub pathways: single nucleotide (SN)-BER, where only one nucleotide
is excised and repaired, and long-patch (LP)-BER. LP-BER occurs when the 5’ residue at the excised
site cannot be processed properly, and more nucleotides need to be removed. This especially occurs
when the lesion is an oxidized base (Robertson, 2009).
NER repairs helix-distorting lesions such as platinum-DNA adducts (Zhu, 2012) and the UVinduced 6-4PPs and CPDs (Palomera-Sanchez, 2011). Like in BER, in NER nucleotides are excised as
well in order to replace them. Differing from BER, in NER a stretch of 24-32 nucleotides among the
lesion is deleted, to ensure the whole area around the DNA adduct is correctly repaired (Kamileri,
2012). I will elaborate on the mechanisms of this pathway in the next chapter.
DNA repair coupled to transcription
During transcription RNA polymerases transcribe from DNA to produce RNA. mRNA can be processed
by ribosomes to gain proteins which function in cellular processes. For transcription of mRNAs, RNA
polymerase II (RNApolII) is required. For transcription to be initiated, RNApolII first needs to bind the
promotor area, to start elongating afterwards. At the promotor RNApolII is first phosphorylated at
Ser-5 by transcription factor II H (TFIIH) to mark an initiating RNA polymerase at the promotor. While
progressing through the gene, the Ser-5 mark declines, and RNA polymerase will be phosphorylated
at Ser-2 to mark the elongating form of RNApolII (Heidemann, 2012).
DNA lesions distorting the DNA double helix pose a challenge for the replication and
transcription machineries. Polymerases might not be able to transcribe or replicate past lesions,
leading to stalled replication forks and RNA polymerases; the processes of transcription and
replication cannot continue while this damage persists. Transcription coupled repair (TCR) is
triggered by stalled RNA polymerases to repair the lesion and ensure a continuation of transcription
afterwards. Transcription coupled repair is a sub pathway of NER, thus also called TC-NER (Hanawalt,
2008). I will elaborate on the mechanisms of TCR in the next chapter.
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NER pathways
Nucleotide excision repair is a
pathway which repairs helix
distorting lesions (Kamileri,
2012). NER can be divided in
two sub pathways, which only
differ in the lesion recognition
step (figure 2). GGR (global
genome repair) is responsible
for the genome wide repair of
small lesions. Transcription
coupled repair (TCR) is
responsible
for
repairing
damage encountered during
RNA transcription on the
transcribed strand (Kamileri,
2012).
After lesion recognition
the TFIIH complex is recruited
to the damaged site and DNA
unwinding starts. Together
with CAK (CDK activating
kinase) and XPG, TFIIH
unwinds the DNA through the
ATP-ase activity of one of its
subunits: XPB (Egly, 2011).
Together with XPA and RPA
(replication protein A), the XPB
and XPD subunits of TFIIH also
Figure 2; Nucleotide excision repair (Hanawalt, 2008) In GGR, lesions
ensure stabilization of the
are recognized by UV-DDB (DDB1 and DDB2) together with XPC-RAD23resulting single stranded DNA
CETN2. In TCR lesions are recognized by the stalling of RNAPOLII, leading
to recruitment of CSB. CSB recruits other NER factors as CSA, p300 ,
(Overmeer,
2011).
For
SAB2, TFIIS, HMGN1 and XPG. After lesion recognition both pathways
excision, ERCC1
(Excision
recruit TFIIH, unwinding the DNA. RPA attaches to the remaining single
repair cross-complementing
stranded DNA for protection while XPF-ERCC1 cuts the DNA. RCF loads
rodent repaid deficiency,
DNA polymerases on the DNA, which are activated by PCNA to fill the
complementation group 1)gap. The final ligation step is performed by a DNA ligase enzyme.
XPF and XPG can be activated
by RPA (Overmeer, 2011). A 24-32-nucleotide fragment around the lesion is cleaved. Following this
step, DNA polymerases are loaded onto the DNA by RFC (replication factor C). The activity of these
polymerases is stimulated by PCNA (Proliferating cell nuclear antigen). The polymerases synthesize
new DNA to fill the gap resulting from the cleaving (Reviewed in Kamileri, 2012). The final product is
ligated together by DNA ligase III-XRCC1 (Moser, 2007) (figure 2).
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Lesion recognition in GGR
In GGR, XPC (xeroderma pigmentosum group C) facilitates lesion recognition together with CETN2
(centrin 2), RAD23 and UV-DDB (UV-damaged DNA-binding protein). Together, these proteins screen
the whole genome for helix distorting DNA damage (Batty, 2000; Nishi, 2005). UV-DDB has ubiquitin
ligase activity due to the DDB2 subunit. However, this ligase activity is sequestered by the inhibitory
action of CSN (Groisman, 2003). After encountering damaged DNA, CSN dissociates from UV-DDB to
remove its inhibitory effect (Groisman, 2003). DDB2 ubiquitinates histones H3 and H4, possibly in
order to release these nucleosomes from the DNA. Releasing the DNA from the nucleosomes makes
bulky DNA adducts within the nucleosomes more accessible to XPC (Wang, 2006). XPC itself is also
ubiquitinated by the DDB2-containing complex to further increase its affinity for DNA (Sugasawa,
2005). Upon DNA recognition, RAD23 dissociates from the complex. At this point the complex TFIIH
(transcription factor II H) is recruited to the lesion site to ensure proper unwinding of DNA and
resulting in further assembly of the repair machinery (Egly, 2011; Kamileri, 2012) (figure 2).
Lesion recognition in TCR
In TCR the DNA damage is recognized by RNApolII; upon encountering helix distorting DNA lesions,
the polymerase is stalled and initiates the DNA repair process (Laine, 2006). Due to the large
footprint of RNApolII stretching over the lesion, the damage site is not accessible for repair proteins.
To ensure accessibility the polymerase needs to move away from the lesion. It has been shown that
this could be done by backtracking of the polymerase to ensure enough space for the binding of
repair factors (Donahue, 1994; Tornaletti, 2001). One of the first proteins to be recruited to the
stalled RNA polymerase is Cockayne syndrome complementation group B (CSB). The recruitment of
CSB to the stalled RNApolII starts the assembly of the whole NER complex (Fousteri, 2006). CSB first
recruits Cockayne syndrome complementation group A (CSA) to the stalled RNA polymerase. CSA and
CSB together recruit the nucleosomal binding protein HMGN1 (high mobility group nucleosome
binding domain 1), XAB2 (XPA binding protein 2) and TFIIS (Fousteri, 2006). As in GGR, the
transcription factor II H (TFIIH) complex is recruited for DNA unwinding (Egly, 2011; Hanawalt, 2008)
(figure 2).
CSB
CSB is very important for TCR: if CSB is depleted or mutated, TCR cannot complete normally, resulting
in Cockayne syndrome, a disease with severe symptoms (Cleaver, 2009). CSB contains an ATP-ase
domain and a ubiquitin-binding domain (UBD). This UBD is necessary for CSB’s function in TCR, since
CSB protein with deleted UBD results in a failure for TCR to complete (Anindya, 2010). Cells
expressing this mutated CSB are able to recruit the whole NER complex, but somehow this complex
does not seem to be functional. The precise mechanism behind this is not yet known (Anindya,
2010).
During the TCR repair process, CSB is ubiquitinated for degradation. It is suggested that
degradation of CSB is necessary for the reinitiation of transcription after the repair of UV damage
(Groisman, 2006). There are a three different E3 ligases suggested to be involved in the
ubiquitination of CSB: BRCA1-BARD1, the p44 subunit of TFIIH and the CSA-Cul4-DDB1 complex
(Groisman, 2006; Takagi, 2005; Wei, 2011).
It seems that next to a function in TC-NER, where CSB is responsible for the recruitment of CSA
and all other NER factors (Fousteri, 2006), CSB might also be involved in other processes in the cell.
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There is evidence indicating that CSB might be involved in both transcription and repair of oxidative
lesions.
CSB loosely interacts with RNApolII during transcription. Upon stalling of the RNA polymerase
due to UV damage, CSB gains a more stable interaction with RNApolII (van Gool, 1997; van den
Boom, 2004). The involvement of CSB in transcription is supported by the fact that CSB has been
shown to be able to alter the chromatin structure and affect transcription in vivo (Newman, 2006).
Compared to normal cells, RNApolII transcription was reduced in undamaged cells lacking CSB and
transcription was stimulated upon expressing CSB in vitro (Dianov, 1997; Selby, 1997). It has also
been suggested that CSB is important for the maintenance of transcription of undamaged genes after
UV-irradiation and oxidative stress (Kyng, 2003; Proietti-De-Santi, 2006). However, the experiments
are still inconclusive; CSB’s role in transcription needs to be studied in more detail.
CSB has been proposed to be active in the repair of oxidative lesions as well, specifically 8-oxoG
and 8-oxoA (Spivak, 2005; Stevnsner, 2008). Cells lacking functional CSB are deficient in the repair of
8-oxoG and 8-oxoA lesions (Dianov, 1999; Tuo, 2002). 8-oxoguanine lesions, shown to be repaired by
CSB, are unlikely to cause a transcriptional arrest, and would thus not be repaired by TC-NER
(Tornaletti, 2004). This indicates that the role of CSB in the repair of oxidative lesions could be
separable from its function in TC-NER, as is possibly the case with CSA as well (Nardo, 2009). This is
supported by the fact that a study from Trapp (2007) showed that repair of oxidative lesions by CSB
can take place in the non-transcribed strand (Trapp, 2007).
CSA
Another important protein for TCR is CSA. Mutations in CSA can cause Cockayne syndrome, similar to
CSB mutations (Cleaver, 2009). CSA associates with DDB1 and Cullin 4A, which contains E3 ubiquitin
activity (Groisman, 2003). Upon UV-damage this complex is recruited to the stalled RNApolII by CSB
(Fousteri, 2006). Upon UV-damage, immediately after CSA recruitment to the site of the lesion, the
CSA-associated ubiquitin ligase activity is silenced by association with COP9 (Constitutive Photo
morphogenesis) (Groisman, 2003). At a later stage in the repair process, the ligase becomes active
again due to dissociation of COP9. It is suggested by Groisman that the CSA-associated ligase activity
is needed at the end of the repair process, and that it functions by degrading repair factors,
specifically CSB, allowing for reinitiation of transcription (Groisman, 2003, 2006).
CSA, next to CSB has also been suggested to have a role in the repair of oxidative damage. Cells
depleted of CSA show sensitivity to oxidative lesions. This role for CSA is separable from its role in
TCR, since a specific mutation in CSA only renders cells sensitive to UV-damage, but not to oxidative
lesions (D’Errico, 2007; Nardo, 2009)
RNApolII regulation in TCR
When encountering a lesion that RNApolII cannot transcribe past, it will stall (Laine, 2006). During
the repair activities the RNA polymerase can backtrack and remain on the DNA (Donahue, 1994;
Tornaletti. 2001). Only if the NER pathway fails to repair the lesion, the polymerase is degraded due
to ubiquitination as a last resort (Wilson, 2012). BRCA1-BARD1 has been suggested to be involved in
the ubiquitination of RNApolII for degradational purposes (Kleiman, 2005; Starita, 2005), although
Anindya and coworkers show that BRCA1-BARD1 depletion does not seem to affect RNApolII
ubiquitination (Anindya, 2007).
The E3 ubiquitin ligase Rsp5 is able to ubiquitinate the largest subunit of RNApolII: Rpb1
(Huibregtse, 1997). Rsp5 has a high preference for Ser2 phosphorylated RNApolII, the elongating
form, but will not bind Ser5 phosphorylated RNApolII, the initiating form of RNApolII (Somesh, 2005).
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In this manner it is ensured that only the elongating form of RNApolII is recognized, and not an RNA
polymerase at the start of an active gene (Wilson, 2012). Rsp5 will add an initial mono-ubiquitin to
Rpb1 that is elongated to a Lys-48 linked ubiquitin chain by the Elc1-Cul3 complex (Harreman, 2009).
A similar process has been suggested in eukaryotes as well where the E3’s NEDD4 and the Elongin
ABC-Rbx1-Cullin 5 complex have a similar interplay. NEDD4 performs a similar role as Rsp5 by
monoubiquitinating Rpb1. The Elongin ABC-Rbx1-Cullin 5 complex elongates the monoubiquitination
of NEDD4 to Lys-48 linked ubiquitin chains at the RPB1 subunit of RNApolII, comparable to the Elc1Cul3 complex (Anindya, 2007; Harreman, 2009). In yeast, The Deubiquitinating enzymes (DUBs) Ubp2
and Ubp3 are found to be able to reverse the ubiquitination steps to rescue the polymerase when
degradation is not necessary (Harreman, 2009). Wilson (2012) suggests a theory explaining why
RNApolII is not degraded while the damage is being repaired by TC-NER. He suggests that this is
caused by a difference in swiftness between the mechanisms. Degradation of RNApolII by the
ubiquitination pathway is much slower than the repair of a lesion found by RNApolII stalling. This
gives more than enough time for the DUBs to reverse the ubiquitination and save the RNA
polymerase from degradation (Wilson, 2012). The induction of the ubiquitination for degradational
purposes is still not entirely clear and more research needs to be done to gain a clear view on the
mechanisms behind RNApolII ubiquitination and its purposes.
Besides ubiquitination for degradation, RNApolII is also ubiquitinated in other ways, potentially
to regulate it in DNA repair. Upon recruitment, Rsp5 also adds Lys-63 linked ubiquitin chains to
RNApolII for regulatory purposes (Harreman, 2009). Another group also showed that BRCA1-BARD1
specifically poly-ubiquitinates the RPB8 subunit of RNApolII, which is not involved in destabilizing
RNApolII (Wu, 2007). Even more, this ubiquitination is specifically found after induction of UVdamage (Wu, 2007). This indicates that ubiquitination of RNApolII as a consequence of UV-damage
has important functions, both for degradational purposes but also for regulatory purposes.
NER related diseases
Defects in the NER pathway can lead to several diseases. Even though the mutations all occur in
proteins in the same pathway, the clinical outcome can vary drastically between patients, depending
on the protein affected and the nature of the mutation (Cleaver, 2009).
Trichothiodystrophy (TTD) is caused by mutations in the subunits XPB, XPD or TTD-A of TFIIH,
active in both the GGR and TCR pathways of NER. Patients are characterized by brittle, sulphur
deficient hair and unusual facial appearances. Further clinical manifestations vary widely from
patients with only brittle hair, to patients with severe developmental defects (Stefanini, 2010).
Xeroderma pigmentosum (XP) patients have mutations in the XP genes (ranging from A to G)
which are active in GGR as well as TCR. Young patients, of only 1 or 2 years of age, suffering from this
disease often show symptoms associated with years of sun exposure, like early extensive freckling.
XP patients have a 2.000 fold increased chance of cancer, including a high incidence of skin cancer.
Patients with neurodegenerative disorders have also been documented (DiGiovanna, 2012).
Cockayne syndrome (CS) is caused by mutations in the TC-NER proteins CSA or CSB. CS patients
suffer from a wide range of developmental and/or progeroid defects and sun sensitivity, but do not
have an increased risk of cancer in contrast to XP patients (Cleaver, 2009).
A less well understood syndrome with TC-NER deficiency is UV-sensitive syndrome (UVSS).
Patients show mild sensitivity for the sun, resulting in acute sunburns, dry skin, freckles, and
sometimes pigmental abnormalities and telangiectasia. They, like CS patients, also do not show any
increased risk for cancer compared to the general population, they actually seem protected from skin
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cancer due to a high apoptosis level in sun-damaged cells combined with a well functioning GGR
system in the remaining cells (Spivak, 2005). However in contrast to CS, UVSSA patients do not show
any developmental defects of neurodegenerative symptoms. Three different complementation
groups of UVSS patients have been identified: patients can have mutations in CSA, CSB or the
previously unidentified gene UVSSA (Nardo, 2009; Spivak, 2005).
When comparing cells of CS and UVSS patients in vitro, there is hardly a difference in response to UV
damage. Both CS and UVSS cells show defective recovery of RNA synthesis after UV-damage. Both
also show UV-sensitivity in colony survival assays due to defective TCR and an accumulation of P53.
GGR is not impaired in either of the cell lines (Spivak, 2005). It seems paradoxical how two diseases
caused by defects in the same DNA repair pathway, TCR, can have such drastically different
symptoms. This phenomenon has in the past been studied by Spivak (2005), and will be elaborated
on in a later chapter by me.
Recently a new protein has been identified to be the causal gene of UVSS when defective: UVsensitive scaffold protein A (UVSSA). UVSSA has been found to interact with the DUB USP7. UVSSA
and USP7 were both found to contribute to the process of TCR and interact with the TCR complex
(Schwertman, 2012; Zhang, 2012). In this review I will try to elucidate the role of UVSSA and USP7 in
this complex through a few studies identifying UVSSA and USP7 as new players in the field. The
finding of UVSSA as a causative gene in UVSS might also help elucidate the different phenotypes in CS
and UVSS.
UVSSA & USP7 in TC-NER
Identification of UVSS-A in UVSSA
UVSSA is a newly discovered protein, identified as the causative gene of UVSS, an unresolved TC-NER
deficiency disorder. UVSSA has been discovered in three different studies, finding the protein by
whole-exome sequencing of UVSS patients (Nakazawa, 2012), screening for altered protein states
upon UV-damage by SILAC (Schwertman, 2012) and a gene complementation screen by microcellmediated transfer (Zhang, 2012). Mutation analysis of UVSS patients revealed that many patients
carried mutations in the UVSSA gene (Nakazawa, 2012; Zhang, 2012). On top of that, knockdown of
UVSSA results in a reduction of recovery of RNA synthesis (RRS), a marker for TCR (Nakazawa, 2012;
Schwertman, 2012). This effect was reversed when wt-tagged UVSSA was transfected both in cells
with si-RNA based knockdown of UVSSA and UVSS-A cells, indicating that UVSSA is involved in TCNER after UV-irradiation, and that it can rescue cells from a UVSS phenotype (Nakazawa, 2012;
Schwertman, 2012; Zhang, 2012). The same effect was not seen on GGR activity, for UV-induced DNA
repair synthesis was not impaired upon UVSSA knockdown, indicating a specific role for UVSSA in TCR
and not GGR (Schwertman, 2012). Together these studies show that UVSSA is a newly discovered
gene in TC-NER, and is causative of UVSS when defective.
Recruitment and interactions
Schwertman and colleagues found that GFP-tagged UVSSA accumulates at local UV-C DNA damage
with similar recruitment kinetics as known TC-NER factors (Schwertman, 2012). UVSSA has been
shown to interact with known TCR factors: subunits of the TFIIH complex (Nakazawa, 2012), CSB and
RNApolII after UV damage (Zhang, 2012). This indicates that UVSSA is involved in TCR at the site of
the UV-damage. Schwertman shows that UVSSA (transiently) binds to RNApolII independent of UVdamage (Schwertman, 2012). They also suggest that UVSSA binds directly to RNApolII and not
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indirectly through CSB, since the same dynamical interaction between RNApolII and UVSSA is found
in CSB depleted cells, as shown by ChIP analysis of in vivo cross-linked cells (Schwertman, 2012).
Zhang et al. propose that both CSA and CSB have an important function in the recruitment of UVSSA
to stalled RNApolII (figure 3B), since in co-IP experiments on a CSA depleted background, UVSSA has
not been found to interact with CSB anymore and the other way around (Fei, 2012; Zhang, 2012).
Zhang et al. also show that UVSSA (transiently) interacts with CSA with and without UV-damage
(Zhang, 2012), proposing that CSA is responsible for the recruitment of UVSSA to CSB. Using life cell
imaging, Schwertman and colleagues however show that UVSSA was still found at the site of DNA
damage in absence of CSA or CSB (Schwertman, 2012). In an IP on cross-linked cells it has been
shown that UVSSA interacts with RNApolII independent of UV-damage, where in a non-cross-linked
setting RNApolII and UVSSA only interacted in response to UV irradiation (Schwertman, 2012; Zhang,
2012). These results suggest that there is always a transient interaction between UVSSA and the
RNApolII complex, but that this interaction is stabilized upon UV damage through CSB and CSA
(Schwertman, 2012; Zhang, 2012).
UVSSA; a function in ubiquitin regulation of TC-NER
UVSSA has both a DUF and a VHS domain. The DUF domain is a domain of unknown function, where
the VHS domain has previously been shown to be implicated in ubiquitin binding (Dicik, 2009). UVSSA
has been found in a screen for proteins with differential ubiquitination upon UV-damage
(Schwertman, 2012). Ubiquitination of UVSSA itself was not increased after UV-damage induction.
Together these data indicate that UVSSA resides in a UV-induced ubiquitinated protein complex
(Schwertman, 2012). Through its VHS domain, UVSSA could potentially bind ubiquitinated RNApolII,
CSB or other ubiquitinated TC-NER factors as a response to UV-damage.
The ubiquitin binding property of UVSSA seems to be important for some of the interactions
between UVSSA and TC-NER factors, since weaker interactions were found between UVSSA and CSB
and TFIIH when UVSSA with mutations in the VHS domain was expressed (Nakazawa, 2012). This
indicates that UVSSA indeed binds TC-NER targets through its ubiquitin binding VHS domain. Even
more, this binding could be essential for UVSSA’s function in TC-NER, since complementing UVSS-A
cells with UVSSA with a mutated VHS domain does not result in normal RRS levels (Nakazawa, 2012).
A protein found to interact with UVSSA is the deubiquitinating enzyme USP7 (Schwertman, 2012;
Zhang, 2012). USP7 has not been linked to TC-NER before, and thus its interaction with UVSSA might
elucidate a new role for this DUB in transcription coupled repair. Knockdown of USP7 has the same
effect on RRS as knockdown of UVSSA, indicating that USP7 indeed plays a role in TC-NER. Since the
interaction between UVSSA and USP7 seems to be independent from UV-damage and chromatin
binding (Zhang, 2012), it has been suggested that the interaction of UVSSA with USP7 forms a stable,
functional complex (Schwertman, 2012; Zhang, 2012). The fact that UVSSA can bind ubiquitinated
targets through its VHS domain and that it interacts with a DUB indicate that UVSSA has an important
role in the ubiquitin regulation of the TC-NER factors.
CSB is ubiquitinated as a response to UV-damage in order to ensure its degradation and facilitate
continuation of transcription after the damage has been repaired (Groisman, 2006). In UVSSA and
USP7 knockdown cells, CSB levels go down upon UV-damage (Fei, 2012; Nakazawa, 2012;
Schwertman, 2012; Zhang, 2012). This drop in CSB levels in itself is not the cause of UVSS-A, since
overexpression of CSB cannot rescue the cells from the effects of UVSSA knockdown (Nakazawa,
2012). This indicates that CSB might be functionally regulated by UVSSA in TCR.
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Schwertman (2012) and Zhang (2012) propose a model where UVSSA is responsible for bringing
USP7 in close vicinity to CSB. USP7 can now facilitate de-ubiquitination of ubiquitinated CSB in the
early stages of repair and rescue it from premature degradation (figure 3A and 3B) (Schwertman,
2012; Zhang, 2012). Schwertman and colleagues suggest that this recruitment of the UVSSA-USP7
complex to CSB is due to binding of UVSSA to RNApolII (Schwertman, 2012) (figure 3A), where Zhang
et al. suggest that this interaction is established by an indirect interaction between UVSSA and
RNApolII through CSA (Zhang, 2012) (figure 3B).
Figure 3; Proposed models for UVSSA involvement in TCR A) Model as proposed by Schwertman et al.
(2012). UVSSA binds directly to RNApolII and brings USP7 close to CSB to keep it in a unubiquitinated state.
B) Model as proposed by Zhang et al. (2012). CSA facilitates the recruitment of UVSSA to RNApolII. USP7,
bound to UVSSA, is now able to keep CSB in a unubiquitinated state. C) Model as proposed by Nakazawa et
al. (2012). UVSSA is recruited to the TCR complex and recruits TFIIH to the stalled RNApolII, which in turn
will ubiquitinated RNApolII. D) Model in absence of UVSSA. When UVSAA is absent, USP7 cannot be brought
close proximity of CSB and CSB will be degraded due to ubiquitination. Also RNApolII is no longer
ubiquitinated and will remain at the damaged site, so no NER complex can form at the lesion site.
Nakazawa and coworkers propose a different model, where UVSSA has a more pronounced
effect on RNApolII. They speculate that UVSSA recruits TFIIH to the site of the lesion to facilitate
ubiquitination of RNApolII. This is supported by an experiment showing that RNApolII is not
ubiquitinated in cells depleted of UVSSA, and that this effect was reversible by expressing UVSSA
cDNA in these cells (Nakazawa, 2012). Nakazawa and colleagues suggest that this ubiquitination of
RNApolII is necessary for its backtracking to allow the formation of the NER machinery at the lesion
site (Nakazawa, 2012) (figure 3C).
In UVSS cells, UVSSA is no longer functionally present. In these cells USP7 can no longer be
brought into close vicinity of CSB, which will result in a reduction of CSB levels, presumably due to
ubiquitin dependent proteasomal degradation (Schwertman, 2012; Zhang, 2012) (figure 3D). Due to
this premature degradation a functional TCR complex cannot form, and transcription will remain
stalled. According to Nakazawa and colleagues, in the absence of UVSSA, RNApolII will not be
ubiquitinated by TFIIH, although Nakazawa et al. propose that there are alternative CSB and CSA
dependent pathways still able to ubiquitinated RNApolII to ensure the same effect, though with a
delay (Nakazawa, 2012) (figure 3D).
P a g e | 13
Proposing a new model for UVSSA and USP7 in TCR
Combining the three studies from
Nakazawa, Schwertman and Zhang with
the already elucidated mechanism of
TCR in literature, many possibilities
arise for the function of UVSSA and
USP7 in TCR. Here I propose one of the
possible models for TCR lesion
recognition regulation with the
incorporation of UVSSA and USP7.
A mechanism for ubiquitination of
RNApolII upon polymerase stalling has
been shown in yeast, and proposed to
work the same in eukaryotes (Anindya,
2007; Harreman, 2009) Upon UVdamage, the E3 ligase complex BRCA1BARD1 can in vivo add polyubiquitin
chains to Rpb8, a subunit of RNApolII
(Wu, 2007). NEDD4 might also be able
to ubiquitinate Rpb1, RNApolII’s largest
subunit, by adding K63 linked ubiquitin
chains (Anindya, 2007). Both UVSSA
and CSB have ubiquitin binding
Figure 4; Proposed model for UVSSA and USP7
domains which have been proven
involvement in TCR A) Upon encountering a lesion and
necessary for proper TC-NER to occur
stalling, RNApolII is poly-ubiquitinated by BRCA1-BARD1 and
(Zhang, 2012; Anindya, 2010).
NEDD4. This ensures a stable binding of the already
The ubiquitin binding VHS domain
transiently interacting proteins CSB and possibly UVSSA.
of UVSSA has been shown to be
CSA, already transiently interacting with UVSSA, is recruited
essential for the interaction of UVSSA
to the site by CSB and might stabilize the whole complex.
with the TCR complex (Zhang, 2012). A
USP7, brought to the complex by UVSSA binding, can
direct interaction between RNApolII
deubiquitinate CSB to prevent its degradation. RNApII is
and UVSSA has been shown
monoubiquitinated by NEDD4, which is polyubiquitinated by
(Schwertman, 2012). It might thus be
Elongin A/B/C-Cullin 4-Rbx2 for a degradational signal. This
polyubiquitin chain could be deubiquitinated by USP7. TFIIH
possible
that
UVSSA
binds
might be recruited to ensure backtracking of RNApolII B) In
ubiquitinated RNApolII through the VHS
the absence of UVSSA, USP7 might not be able protect CSB
domain. This interaction could function
and RNApolII from proteosomal degradation, and no NER
to stabilize an already transient
complex might form to repair the lesion.
interaction between UVSSA and
RNApolII, since UVSSA was found to transiently interact with RNApolII, even without damage
(Schwertman, 2012). Due to the preformed complex of USP7 and UVSSA, USP7 is also recruited to
the stalled, ubiquitinated RNApolII.
The ubiquitin binding domain of CSB has been shown not to be important for assembly of the
TC-NER complex, but is important for the functioning of this complex (Anindya, 2010). It could thus
14 | P a g e
be the case that CSB binds RNApolII upon stalling but that the whole TC-NER complex only becomes
functional when the UBD domain of CSB interacts with ubiquitinated RNApolII. When UVSSA and CSB
are both recruited to the site of damage, CSB additionally recruits CSA (Fousteri, 2006). CSA could
stabilize the interaction between UVSSA and RNApolII since it was shown that CSA has a role in the
interaction between UVSSA and CSB (Zhang, 2012). CSB might inadvertently be ubiquitinated by
BRCA1-BARD1, CSA-Cul4-DDB1 or the p44 subunit of TFIIH (Groisman, 2006; Takagi, 2005; Wei,
2011), even though ubiquitination due to CSA’s ubiquitin ligase activity should be prevented by the
inhibition of COP9 (Groisman, 2003). USP7 is now close enough to CSB to facilitate its
deubiquitinating activity and rescue it from premature degradation prior to the recruitment of NER
complex factors. In this way UVSSA together with USP7 can ensure the correct formation of a NER
complex to facilitate repair of the lesion.
The E3 NEDD4 also mono-ubiquitinates RNApolII, which can then be polyubiquitinated by the
Elongin ABC-Rbx1-Cullin 5 complex to K48 linked ubiquitin chains (Harreman, 2009). This
polyubiquitination is a signal for degradation. RNApolII needs to be protected from this signal to
prevent loss of already transcribed RNA. It is possible that the DUB activity of USP7 functions to
deubiquitinate the K48-linked ubiquitin chain to prevent the degradation of RNApolII while the TCR
machinery is still working on the lesion.
Implementing Nakazawa’s model into this, it is possible that UVSSA also recruits TFIIH to the site.
The p44 subunit of TFIIH itself also has E3 ligase activity, which could result in the ubiquitination of
RNApolII. This ubiquitin mark could be responsible for the backtracking of RNApolII, as proposed by
Nakazawa et al. (Nakazawa, 2012).
When a cell is UVSSA deficient, USP7 cannot save CSB or RNApolII from ubiquitin dependent
degradation, nor can it recruit TFIIH (Fei, 2012; Nakazawa, 2012; Schwertman, 2012; Zhang, 2012).
If USP7 indeed saves CSB from degradation, then in UVSSA deficient cells there will be errors in
the assembly of the rest of the TCR machinery, including a failure to recruit TFIIH due to premature
degradation of CSB.
If, on the other hand, USP7 functions to deubiquitinate K48 linked ubiquitin chains on RNApolII
to save it from degradation, lack of UVSSA might result in degradation of RNApolII. This degradation
of RNApolII could explain why RNApolII does not return 8 hours after UV-irradiation in UVSS-A cells,
in contrast to cells normally expressing UVSSA (Schwertman, 2012, Zhang, 2012). The subsequent
delay in RRS can also be explained by this phenomenon (Schwertman, 2012, Nakazawa, 2012).
Another consequence of lack of functional UVSSA might be that RNApolII ubiquitination is
reduced due to the indirect effect of UVSSA on TFIIH recruitment. Decrease of this ubiquitination
might lead to a failure in the backtracking of RNApolII (Nakazawa, 2012). Consequently, the TC-NER
complex cannot reach the damaged site, and repair cannot take place.
Implications on Cockayne syndrome vs. UV-sensitive syndrome
In this model UVSSA positively regulates CSB: when UVSSA is absent in the cells, CSB is also not able
to function in TC-NER. Since UVSSA affects the stability of CSB, you would expect that UVSSA
depletion would give a similar or worse, phenotype than defects in CSB. Even though, patients with
defective mutations in CSB generally gain a much more severe phenotype than patients with a loss of
UVSSA (Spivak, 2005).
When considering the precise functions of both proteins there might be an explanation for this
phenomenon. CSB has been shown to have more functions in the cell then only in TC-NER. It has
been shown to be active in repair of oxidative lesions (Dianov, 1999; Spivak, 2005; Stevnsner, 2008;
P a g e | 15
Tuo, 2002) and perhaps even transcription (Dianov, 1997; Kyng, 2003; Newman, 2006; Proietti-DeSanti, 2006; Selby, 1997), where UVSSA so far has only been shown to have a role in TC-NER. This
indicates that mutations in UVSSA only cause a misregulation of CSB in TC-NER, but does not disrupt
other functions of CSB. Mutations in CSB could disrupt all functions of CSB.
There is ample evidence that CSB plays a role in the repair of oxidative lesions (Trapp, 2007). It
has been shown that cells from CS patients have a higher sensitivity to oxidative lesions than cells
from UVSS patients (D’Errico, 2007; Nardo, 2009; Spivak, 2006), indicating that CS patients might
indeed carry mutations in CSA or CSB which affects the repair of oxidative lesions. There are
implications that the function of CSB and CSA in the repair of oxidative lesions might be the cause of
the neurodegenerative symptoms in CS. Neural tissue has a higher level of oxidative metabolism then
other tissues, increasing the sensitivity of neuronal tissue to oxidative lesions, especially in defects in
the repair of these lesions (Cleaver, 2009). Also, brains of CS patients show accumulations of
oxidative DNA damage in the neurons, possibly being associated with the neurodegenerative
phenotype (Hayashi, 2005). During early development, the active metabolism generates high levels
of oxidative lesions (Hanawalt, 2008), this could also lead to developmental defects as seen in CS
patients.
A role for CSB in transcription has also been hypothesized. CSB has been shown to be important
for RNApolII dependent transcription, maintenance of the transcription of undamaged genes and
altering the chromatin structure, affecting transcription (Dianov, 1997; Kyng, 2003; Newman, 2006;
Proietti-De-Santi, 2006; Selby, 1997). Even though more research needs to be done to conclusively
prove CSB’s role in transcription separately from its function in TC-NER, it is possible that defects in
transcription next to defective DNA repair might explain the developmental defects in CS (Kamileri,
2012).
Since cells from UVSS patients do not show sensitivity to oxidative lesions, it is likely that the
symptoms in UVSS are only caused by a UV-sensitivity due to loss of UVSSA, solely misregulating
CSB’s function in TCR, keeping CSB intact to fulfill its other cellular functions. CS symptoms on the
other hand are caused by mutations in CSA or CSB. These mutations can lead to a complete loss of
the protein or completely dysfunctional proteins. This could result in a defect in the repair of
oxidative lesions and/or transcription next to a defect in TC-NER due to a broader loss of CSB
function, possibly explaining the UV-sensitivity, the neurodegeneration as well as the developmental
defects.
These hypotheses do not explain the case where a mutation in CSB results in a complete loss of CSB
but only gives rise to UVSS and not CS (Horibata, 2004). If CSB is completely lost to the cell, it cannot
function in any process. However, there are also patients known who are not able to produce a CSB
protein, who do show severe clinical symptoms (Laugel, 2008). Patients with the exact same
mutations in CSB can have different clinical manifestations (Colella, 2000). It seems that the nature of
the mutation does not always exactly predict the outcome of the disease. These mutations can thus
not be the sole cause of the symptoms. Cleaver suggests that the case where there is no CSB product
found due to a termination mutation might still express CSB in low levels, enough to handle oxidative
lesions properly, though not enough to ensure correct TC-NER (Cleaver, 2009). He also proposes that
the other variations in symptoms between patients with identical mutations might be due to a
different internal status of their reactive oxygen species (Cleaver, 2009).
16 | P a g e
It is interesting to note that a conserved fusion-protein of CSB has been found. It entails a version
where the piggyBac transposable element PGBD3 has integrated into the 5th intron of the CSB gene,
resulting in 3 different protein products: fully intact CSB, a protein where PGBD3 is fused to the first
five exons from CSB and one with only the PGBD3 transposase (Bailey, 2012; Newman, 2008). This
fusion CSB-PGBD3 fusion protein pushes the repair of UV-damage over the repair of oxidative
lesions, even in the absence of functional CSB (Bailey, 2012).
This could indicate that mutations affecting CSB’s ability to repair oxidative damage might be
located somewhere after the 5th intron, where the first 5 introns mainly participate in the function of
CSB in TCR. When mapping mutations in CS patients however, there are many patients with
mutations in the first 5 introns of CSB that do not always cause depletion of the CSB protein and do
show severe CS symptoms (Laugel, 2010).
It has been suggested by Newman (2008) that this piggyBac fusion protein might cause the CS
phenotype in the absence of functional full length CSB. This however does not make sense in the
light of mutation sites in CSB causing CS; (truncating) mutations are found both 3’ and 5’ of the
transposon insertion, also preventing expression of the fusion protein when the mutation is found
primal to the insertion site. Also, the presence of this fusion protein is not correlated to the severity
of the CS symptoms, rendering it highly unlikely that it is the causative gene of CS (Cleaver, 2009).
Concluding remarks
Even though the exact mechanism behind Cockayne syndrome cannot be fully explained yet, the
finding of a protein complex specifically active in TCR and not any other processes is another step
forward in explaining the difference in UVSS and CS. More research needs to be done to gain more
insight in the pathway of TCR though. It is vital to gain more insight in the ubiquitination mechanisms
in TCR. First of all it is still unclear which E3 ligases are responsible for the ubiquitination of the
different targets like CSB and RNApolII. Also, even though the recent experiments already elucidated
much on the functions of UVSSA and UPS7 in TCR, the exact functions of these proteins need to be
further established. For example, it needs to be verified whether UVSSA binds to a specific
ubiquitinated domain through its VHS domain, and which domain of which protein this is. Further, it
needs to be verified if USP7’s DUB activity only deubiquitinates CSB, or also other proteins in the TCR
complex. In addition, more experiments need to be done to further explain the process behind the
symptoms of Cockayne syndrome. It is interesting to look more closely to the repair of oxidative
lesions in this setting to try to understand the involvement of this process in CS. The role of CSB and
other TC-NER factors in transcription might also be interesting to clarify to gain a better
understanding of the Cockayne syndrome symptoms possibly more related to transcriptional
repression.
Since cells from UVSS patients and CS patients hardly show a difference in vitro as a response to
UV-damage, making it hard to elucidate the exact difference between the two diseases, it might be
interesting to look at a more in vivo setting. Developing mouse models carrying the mutations
causing the different diseases can give more insight in the in vivo differences between UVSS and CS.
Even though mouse models carrying mutations in CSA and CSB have been developed, they show a
phenotype which does not correspond to CS as seen in humans. When inactivating the GGR in these
mice a more comparable model for human CS can be obtained (Kamileri, 2012). Mouse models for
UVSS are momentarily being developed in the lab of Vermeulen (personal communication,
Schwertman, 2012).
P a g e | 17
Acknowledgements
First of all, I would like to thank Petra Schwertman for her excellent guidance during the writing of
this thesis. She was always there to help me when I got stuck or did not understand some things. She
always had time to help me, and was able to point out just the things to help me improve my writing
and understanding of the subject. I learned a lot from her during the process of writing this thesis.
Further I greatly appreciate the help of Luuk Bevers by proofreading my manuscript, and for keeping
me focused on my work. I also am very grateful to Jurgen Marteijn and Puck Knipscheer for
evaluating my thesis.
18 | P a g e
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