14 research outputs found
Focusing on the Versatile Transcription-Coupled DNA Repair Pathway
Many aspects of TC-NER have been described since the discovery of this
versatile DNA damage repair pathway three decades ago [123]. However,
many crucial questions regarding its exact molecular mechanism and the
manner in which it deals with different types of lesions remain
unanswered [31].
To further unravel the TC-NER mechanism, sensitive techniques that can
specifically measure TC-NER activity would be of great value. In Chapter 2
the development of a new, single-cell assay that can quantify TC-NER
activity is described. This immunofluorescence-based method allows the
direct measurement of TC-NER activity in an user-friendly manner.
Furthermore, this sensitive assay not only enables the measurements of
TC-NER and GG-NER activity on low, physiological relevant, UV-C doses (2
J/m2), but also allows detection and quantification of the activity of other
excision repair pathways.
Thus far, the exact mechanism how UVSSA is recruited to the TC-NER
complex remains elusive. Therefore, we studied the accumulation of
UVSSA on UV-C induced DNA damage in Chapter 3. Using live cell
microscopy, we showed that UVSSA is recruited to DNA damage in a CSA
and CSB independent manner. We further showed, using specific UVSSA
deletion mutants that the DUF2043 domain is important for its
recruitment to UV-induced DNA damage. To identify factors involved in
the recruitment of UVSSA to DNA damage, a quantitative mass
spectrometry approach was used to reveal proteins that specifically
interact with the DUF2043 domain. With this approach we identified the
FACT subunit Spt16 as a novel UVSSA interactor and follow-up studies
indicated that Spt16 is involved in the recruitment of UVSSA to sites of
DNA damage.
As UVSSA is hypothesised to be involved in the response to both UV and
oxidative induced DNA damage, in Chapter 4 we used quantitative
interaction proteomics to identify UVSSA interactions that were
specifically induced following UV-C or H2O2 induced DNA damage. In this
chapter we describe the damage-specific UVSSA interaction partners,
discuss their potential roles and propose that UVSSA might have different
functions following UV or oxidative DNA damage.
In Chapter 5, the function of the TC-NER factor CSB during the repair of
oxidative damage was analysed. Live cell imaging studies indicated that
the recruitment of XRCC1 to oxidative lesions is dependent on functional
CSB and active transcription, whereas recruitment of the BER-initiating
glycosylase OGG1 does not require transcription or CSB. Based on our data
we propose a model in which CSB facilitates XRCC1 recruitment to RNA
polymerase II complexes stalled at BER-intermediates. These results
further establish the importance of CSB in BER.
In Chapter 6 we discuss the main findings of the experimental work
described in this thesis and provide future directions to study the role and
molecular function of TC-NER factors in the repair of different types of
DNA damage
The transcription-coupled DNA repair-initiating protein CSB promotes XRCC1 recruitment to oxidative DNA damage
Transcription-coupled nucleotide excision repair factor Cockayne syndrome protein B (CSB) was suggested to function in the repair of oxidative DNA damage. However thus far, no clear role for CSB in base excision repair (BER), the dedicated pathway to remove abundant oxidative DNA damage, could be established. Using live cell imaging with a laser-assisted procedure to locally induce 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, we previously showed that CSB is recruited to these lesions in a transcription-dependent but NER-independent fashion. Here we showed that recruitment of the preferred 8-oxoG-glycosylase 1 (OGG1) is independent of CSB or active transcription. In contrast, recruitment of the BER-scaffolding protein, X-ray repair cross-complementing protein 1 (XRCC1), to 8-oxoG lesions is stimulated by CSB and transcription. Remarkably, recruitment of XRCC1 to BER-unrelated single strand breaks (SSBs) does not require CSB or transcription. Together, our results suggest a specific transcription-dependent role for CSB in recruiting XRCC1 to BER-generated SSBs, whereas XRCC1 recruitment to SSBs generated independently of BER relies predominantly on PARP activation. Based on our results, we propose a model in which CSB plays a role in facilitating BER progression at transcribed genes, probably to allow XRCC1 recruitment to BER-intermediates masked by RNA polymerase II complexes stalled at these intermediates
FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER
Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway
Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair
textabstractNucleotide excision repair (NER) comprises two damage recognition pathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which remove a wide variety of helix-distorting lesions including UV-induced damage. During NER, a short stretch of single-stranded DNA containing damage is excised and the resulting gap is filled by DNA synthesis in a process called unscheduled DNA synthesis (UDS). UDS is measured by quantifying the incorporation of nucleotide analogues into repair patches to provide a measure of NER activity. However, this assay is unable to quantitatively determine TC-NER activity due to the low contribution of TCNER to the overall NER activity. Therefore, we developed a user-friendly, fluorescence-based single-cell assay to measure TC-NER activity. We combined the UDS assay with tyramide-based signal amplification to greatly increase the UDS signal, thereby allowing UDS to be quantified at low UV doses, as well as DNA-repair synthesis of other excision-based repair mechanisms such as base excision repair and mismatch repair. Importantly, we demonstrated that the amplified UDS is sufficiently sensitive to quantify TCNER-derived repair synthesis in GG-NER-deficient cells. This assay is important as a diagnostic tool for NER-related disorders and as a research tool for obtaining new insights into the mechanism and regulation of excision repair
The transcription-coupled DNA repair-initiating protein CSB promotes XRCC1 recruitment to oxidative DNA damage
International audienceTranscription-coupled nucleotide excision repair factor Cockayne syndrome protein B (CSB) was suggested to function in the repair of oxidative DNA damage. However thus far, no clear role for CSB in base excision repair (BER), the dedicated pathway to remove abundant oxidative DNA damage, could be established. Using live cell imaging with a laser-assisted procedure to locally induce 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, we previously showed that CSB is recruited to these lesions in a transcription-dependent but NER-independent fashion. Here we showed that recruitment of the preferred 8-oxoG-glycosylase 1 (OGG1) is independent of CSB or active transcription. In contrast, recruitment of the BER-scaffolding protein, X-ray repair cross-complementing protein 1 (XRCC1), to 8-oxoG lesions is stimulated by CSB and transcription. Remarkably , recruitment of XRCC1 to BER-unrelated single strand breaks (SSBs) does not require CSB or transcription. Together, our results suggest a specific transcription-dependent role for CSB in recruiting XRCC1 to BER-generated SSBs, whereas XRCC1 recruitment to SSBs generated independently of BER relies predominantly on PARP activation. Based on our results, we propose a model in which CSB plays a role in facilitating BER progression at transcribed genes, probably to allow XRCC1 recruitment to BER-intermediates masked by RNA polymerase II complexes stalled at these intermediates
Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair
Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II
A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair
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