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

Baalbek, Libanon. Die Arbeiten des Jahres 2019

Field research in Baalbek, located in the northern Beqaa plain of Lebanon, was resumed in summer 2019. Investigations took place in three areas and scholarly topics: small soundings in an area with medieval and late antique architectural remains should evaluate the scientific potential. In the famous Jupiter sanctuary, systematic investigation was also carried out into the use of colours in the buildings. In addition, the excavations in the pre-Roman mound under the altar courtyard of the Jupiter sanctuary were resumed and Middle Bronze Age strata investigated. The planing for the conservation and presentation of the ruins were also continued. Projects for the presentation of the monumental Roman bath are currently  being developed

Baalbek, Libanon. Feldforschungen und Konservierungsarbeiten. Die Arbeiten des Jahres 2020

In Baalbek, the development of the city from a prehistoric tell to a Roman city and its constant metamorphosis into the early modern period has been the subject of research for many years. Adapting to travel and work oppor­tunities and taking into account the COVID-19 pandemic, fieldwork in Baalbek also took place in 2020, but in several working stages and for a shorter period than planned. Work continued in three areas: with photographic and photogrammetric documentation in the Roman sanctuary of Jupiter, with exca­vations in the medieval quarter in the so-called Bustan Nassif, and for the preparation of conservation measures in the ›Bustan el-Khan‹ with its public buildings from the Roman period

Middle to Late Holocene Variations in Salinity and Primary Productivity in the Central Baltic Sea: A Multiproxy Study From the Landsort Deep

Anthropogenic forcing has led to an increased extent of hypoxic bottom areas in the Baltic Sea during recent decades. The Baltic Sea ecosystem is naturally prone to the development of hypoxic conditions due to its geographical, hydrographical, geological, and climate features. Besides the current spreading of hypoxia, the Baltic Sea has experienced two extensive periods of hypoxic conditions during the Holocene, caused by changing climate conditions during the Holocene Thermal Maximum (HTM; 8–4.8 cal ka BP) and the Medieval Climate Anomaly (MCA; 1–0.7 cal ka BP). We studied the variations in surface and bottom water salinity and primary productivity and their relative importance for the development and termination of hypoxia by using microfossil and geochemical data from a sediment core retrieved from the Landsort Deep during IODP Expedition 347 (Site M0063). Our findings demonstrate that increased salinity was of major importance for the development of hypoxic conditions during the HTM. In contrast, we could not clearly relate the termination of this hypoxic period to salinity changes. The reconstructed high primary productivity associated with the hypoxic period during the MCA is not accompanied by considerable increases in salinity. Our proxies for salinity show a decreasing trend before, during and after the MCA. Therefore, we suggest that this period of hypoxia is primarily driven by increasing temperatures due to the warmer climate. These results highlight the importance of natural climate driven changes in salinity and primary productivity for the development of hypoxia during a warming climate

Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants

DNA methylation, an essential regulator of transcription and chromatin structure, is established and maintained by the coordinated action of three DNA methyltransferases: DNMT1, DNMT3A and DNMT3B, and the inactive accessory factor DNMT3L. Disruptions in DNMT3B function are linked to carcinogenesis and genetic disease. DNMT3B is also highly alternatively spliced in a tissue- and disease-specific manner. The impact of intra-DNMT3 interactions and alternative splicing on the function of DNMT3 family members remains unclear. In the present work, we focused on DNMT3B. Using a panel of in vitro assays, we examined the consequences of DNMT3B splicing and mutations on its ability to bind DNA, interact with itself and other DNMT3's, and methylate DNA. Our results show that, while the C-terminal catalytic domain is critical for most DNMT3B functions, parts of the N-terminal region, including the PWWP domain, are also important. Alternative splicing and domain deletions also influence DNMT3B’s cellular localization. Furthermore, our data reveal the existence of extensive DNMT3B self-interactions that differentially impact on its activity. Finally, we show that catalytically inactive isoforms of DNMT3B are capable of modulating the activity of DNMT3A–DNMT3L complexes. Our studies therefore suggest that seemingly ‘inactive’ DNMT3B isoforms may influence genomic methylation patterns in vivo

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

Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms