Spotting DNA damage repair pathways: Real-time methods and mechanisms

DNA is the molecule that carries the genetic information within a cell, but its integrity is constantly challenged by multiple endogenous and exogenous sources. Damage in the DNA can perturb essential processes such as transcription or replication. If the DNA damage is left unrepaired, it can result in mutation accumulation which can lead to tumorigenesis, and/or senescence and apoptosis, which can accelerate aging and cause neurodegeneration 0n an organismal level. Therefore, cells are equipped with multiple DNA repair mechanisms that help them deal with numerous types of DNA damage.
In Chapter 1, we introduce these DNA repair mechanisms with special attention on Double-Strand Break (DSB) repair, nucleotide excision Repair (NER) and Interstrand Crosslink Repair (ICLR).
Since DNA is tightly packed, chromatin remodelers are essential to increase chromatin accessibility and regulate the accumulation of repair proteins at the site of DNA damage. One of such chromatin remodelers is the SWI/SNF family, which is frequently mutated in cancer. SWI/SNF has been previously implicated in several DNA repair pathways, but its roles and regulation remain unclear. In Chapter 2, we study the multiple functions of SWI/SNF family of chromatin remodelers during Transcription-Coupled Homologous Recombination (TC-HR), an HR subpathway that takes place to repair DSBs in actively transcribing genes. Here, we show that multiple SWI/SNF complexes coordinately perform different roles during TC-HR. First, SWI/SNF helps in the accumulation of RAD52 to promote DNA repair. Secondly, SWI/SNF promotes R-loop resolution. Third, SWI/SNF is actively involved in transcriptional silencing by promoting RNA polymerase II eviction and maintenance of the silencing over time.
It has always been intriguing why different mutations in NER encoding genes lead to different syndromes. In Chapter 3, we investigated the phenotypes caused by deficiencies in the nucleases XPF and XPG and, compared them with deficiencies in other NER core proteins such as XPA or TTDA. We demonstrate that, in the absence of the nucleases, the repair intermediates are stalled at the lesion shielding it from other repair mechanisms. Thus, by destabilizing the TFIIH binding to the lesion we could mildly alleviate the stronger phenotypes observed in the absence of XPF or XPG. This helped us further understand why the consequences of not having functional nucleases during NER cause increased severity in the phenotypes.
In Chapter 4, we develop a novel technique to study DNA repair efficiency. Traditionally, since transcription is silenced after DNA damage, studying transcription restart was used to measure repair. Here, we use translation restart as a redout for DNA repair efficiency. We demonstrate that this method is sensitive and reproducible and can be used both in vitro and in vivo systems. Moreover, there are multiple ways in which this technology can be used, making it suitable for real-time analysis and diagnostics.
In Chapter 5 we identify UBR5 E3-ubiquitin ligase as a novel XPF interactor. We describe that UBR5 affects the levels of XPF-ERCC1 complex and its ICL-specific interactor SLX4. By using live cell confocal imaging, we find that XPF recruitment to both NER and ICLR is compromised in the absence of UBR5. To investigate whether UBR5 has a function at the site of damage, we also followed its accumulation to both NER and ICLR and found that it was not actively recruited to DNA damage, suggesting that it has an indirect role in regulating XPF-ERCC1- SLX4 function. Moreover, by using CRISPR/Cas9, we generated UBR5 knockout cell lines that will be used to find UBR5 unknown targets and possibly understand its role in DNA repair.
Finally, Chapter 6 the contents of this thesis are summarized and discussed. We suggest future research plans to further understand the details and regulation of the findings described in this thesis.