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.