Ubiquitin-Mediated Regulation of Damage Recognition in Nucleotide Excision Repair

__Abstract__ __Scope of the thesis:__ The integrity of our genetic information is continuously threatened by endogenous metabolites and environmental agents that can generate a variety of DNA lesions. Accumulation of DNA damage can induce genetic changes or cell death, which may result in the onset of cancer or premature ageing. To deal with these adverse effects a network of DNA repair mechanisms and damage signaling pathways, known as the DNA Damage Response (DDR), has evolved. Nucleotide Excision Repair (NER) is responsible for the repair of a wide variety of helix distorting lesions, including those induced by UV-light. NER is a multistep process, which requires the action of more than 30 proteins that need to be tightly controlled to function at the right time and place to warrant efficient repair. Complex cellular processes are commonly regulated by various post translational modifications. Most notably, protein ubiquitylation has emerged as a key regulator of NER. The aim of the work presented in this thesis is to understand the regulation and dynamic properties of NER factors and the UV-DDR in general by ubiquitylation. Chapter 1 provides the necessary background and the current knowledge on ubiquitinmediated regulation of the DNA damage recognition steps of NER. Mass spectrometry (MS) can be used to study the ubiquitylation status of proteins on a proteome wide scale. Since ubiquitylation is transient and solely occurs on a small fraction of proteins, methods to enrich for these proteins are required to study them with MS. In chapter 2, a quantitative proteomics approach was combined with the isolation of ubiquitylated peptides to identify UV-regulated ubiquitylation sites on proteins. In addition to the well-known ubiquitylated NER factors, XPC and DDB, we identified UV-responsive ubiquitylated proteins that are active in different biological pathways including, DNA repair, chromatin remodeling, transcription, mRNA splicing, translation and the ubiquitin proteasome system. The most UVinduced peptides were identified for Histone H1. UV-induced H1 ubiquitylation was validated by biochemical experiments. Chapter 3 describes the identification and characterization of a new ubiquitin E3 ligase - RNF111 - required for efficient NER. RNF111 belongs to the class of SUMO-targeted ubiquitin ligases (STUbLs), which provide direct crosstalk between SUMOylation and ubiquitylation. RNF111 specifically recognizes proteins modified with poly-SUMO2/3 chains, and promotes UBC13-dependent K63-linked ubiquitylation. We demonstrate that RNF111 targets SUMOylated XPC, a DNA damage recognition factor in NER. In chapter 4 we have studied the function of the RNF111 mediated XPC ubiquitylation in vivo. Using a combination of DNA repair assays, immunofluorescence and live cell imaging experiments, we show that RNF111-mediated ubiquitylation stimulates the release of XPC from DNA lesions. This step is required for the stable incorporation of the NER endonucleases XPG and ERCC1/XPF to efficiently complete the NER reaction. In chapter 5 we further focus on XPC dynamics. In contrast to other NER factors, XPC shows a non-linear immobilization in response to increasing UV-doses. XPC binding is inhibited at low UV-C doses (0-4 J/m2), which is dependent on Cul4a and XPC ubiquitylation. NER comprises two damage recognition sub-pathways: global genome NER (GG-NER), involving XPC, and transcription coupled NER (TC-NER). We propose a model in which cells switch between suppression of stable binding of XPC to DNA lesions at low UV-doses and release of this inhibition at higher doses. This bimodal switch allows cells to prioritize repair of transcription blocking DNA lesions under mild genotoxic stress (low UV-dose, ≤ 4 J/m2). Chapter 6 describes the dynamic behavior of RPA in replication and NER. In contrast to other replication factors, RPA does not cluster in replication foci due to a very short residence time at single stranded DNA. During NER, RPA is involved in both the pre- and post-incision steps of NER. RPA binding to the pre-incision complex could only be visualized in the absence of incision without a substantial increase in residence time. Our data show that RPA is an intrinsically highly dynamic protein. In chapter 7 the main findings of this thesis are wrapped up and the perspectives derived from these data to obtain a more in depth view on the regulation of NER are being discussed

DNA damage-induced histone H1 ubiquitylation is mediated by HUWE1 and stimulates the RNF8-RNF168 pathway

The DNA damage response (DDR), comprising distinct repair and signalling pathways, safeguards genomic integrity. Protein ubiquitylation is an important regulatory mechanism of the DDR. To study its role in the UV-induced DDR, we characterized changes in protein ubiquitylation following DNA damage using quantitative di-Gly proteomics. Interestingly, we identified multiple sites of histone H1 that are ubiquitylated upon UV-damage. We show that UV-dependent histone H1 ubiquitylation at multiple lysines is mediated by the E3-ligase HUWE1. Recently, it was shown that poly-ubiquitylated histone H1 is an important signalling intermediate in the double strand break response. This poly-ubiquitylation is dependent on RNF8 and Ubc13 which extend pre-existi

Characterization of the mammalian family of DCN-type NEDD8 E3 ligases

Cullin-RING ligases (CRL) are ubiquitin E3s that bind substrates through variable substrate-receptor proteins. CRLs are activated by attachment of the ubiquitin-like protein NEDD8 to the Cullin subunit and DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like proteins and little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and Cullins that can only be neddylated in the presence of substrate adaptor. These DCNL-CUL-CAND1 complexes may represent “reserve” CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most Cullin subtypes, but that they are likely responsible for the neddylation of different subpopulations of any given Cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their Cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression

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

UCHL3 Regulates Topoisomerase-Induced Chromosomal Break Repair by Controlling TDP1 Proteostasis

Genomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair

Protection from ultraviolet damage and photocarcinogenesis by vitamin d compounds

© Springer Nature Switzerland AG 2020. Exposure of skin cells to UV radiation results in DNA damage, which if inadequately repaired, may cause mutations. UV-induced DNA damage and reactive oxygen and nitrogen species also cause local and systemic suppression of the adaptive immune system. Together, these changes underpin the development of skin tumours. The hormone derived from vitamin D, calcitriol (1,25-dihydroxyvitamin D3) and other related compounds, working via the vitamin D receptor and at least in part through endoplasmic reticulum protein 57 (ERp57), reduce cyclobutane pyrimidine dimers and oxidative DNA damage in keratinocytes and other skin cell types after UV. Calcitriol and related compounds enhance DNA repair in keratinocytes, in part through decreased reactive oxygen species, increased p53 expression and/or activation, increased repair proteins and increased energy availability in the cell when calcitriol is present after UV exposure. There is mitochondrial damage in keratinocytes after UV. In the presence of calcitriol, but not vehicle, glycolysis is increased after UV, along with increased energy-conserving autophagy and changes consistent with enhanced mitophagy. Reduced DNA damage and reduced ROS/RNS should help reduce UV-induced immune suppression. Reduced UV immune suppression is observed after topical treatment with calcitriol and related compounds in hairless mice. These protective effects of calcitriol and related compounds presumably contribute to the observed reduction in skin tumour formation in mice after chronic exposure to UV followed by topical post-irradiation treatment with calcitriol and some, though not all, related compounds

Mission Critical Communication

In response to the growing demands on emergency medical services, this project focused on enhancing communication tools to improve patient outcomes. By collaborating with Jabra and the Umeå Ambulance station, I’ve aimed to leverage advanced communication technology for more efficient and effective emergency care. The result is Jabra Assist, a high-bandwidth communication device that incorporates live video streaming. It is targeted at completely replacing the traditional hand-held radio, anticipating the possibilities unlocked by a transition from radio to wireless broadband infrastructure for emergency services.Paramedics wear the device on the chest, allowing remote medical specialists to assist and guide them in real time while they work with the patient. This method of remote assistance has been found to result in more accurate assessments, enhanced treatment capabilities, and fewer unnecessary hospital transports, saving precious emergency room capacity for those who truly need it

Mission Critical Communication

In response to the growing demands on emergency medical services, this project focused on enhancing communication tools to improve patient outcomes. By collaborating with Jabra and the Umeå Ambulance station, I’ve aimed to leverage advanced communication technology for more efficient and effective emergency care. The result is Jabra Assist, a high-bandwidth communication device that incorporates live video streaming. It is targeted at completely replacing the traditional hand-held radio, anticipating the possibilities unlocked by a transition from radio to wireless broadband infrastructure for emergency services.Paramedics wear the device on the chest, allowing remote medical specialists to assist and guide them in real time while they work with the patient. This method of remote assistance has been found to result in more accurate assessments, enhanced treatment capabilities, and fewer unnecessary hospital transports, saving precious emergency room capacity for those who truly need it