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.

Histone acetylation influences protein interactions and chromatin accessibility and plays an important role in the regulation of transcription, replication, and DNA repair. Conversely, DNA damage affects these crucial cellular processes and induces changes in histone acetylation. However, a comprehensive overview of the effects of DNA damage on the histone acetylation landscape is currently lacking. To quantify changes in histone acetylation, we developed an unbiased quantitative mass spectrometry analysis on affinity-purified acetylated histone peptides, generated by differential parallel proteolysis. We identify a large number of histone acetylation sites and observe an overall reduction of acetylated histone residues in response to DNA damage, indicative of a histone-wide loss of acetyl modifications. This decrease is mainly caused by DNA damage-induced replication stress coupled to specific proteasome-dependent loss of acetylated histones. Strikingly, this degradation of acetylated histones is independent of ubiquitylation but requires the PA200-proteasome activator, a complex that specifically targets acetylated histones for degradation. The uncovered replication stress-induced degradation of acetylated histones represents an important chromatin-modifying response to cope with replication stress.

Nucleotide excision repair (NER) plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR) mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will discuss current knowledge gained from the use of C. elegans to study NER and the response to UV-induced DNA damage.

Protein-protein interactions regulate many essential enzymatic processes in the cell. Somatic mutations outside of an enzyme active site can therefore impact cellular function by disruption of critical protein-protein interactions. In our investigation of the cellular impact of the T304I cancer mutation of DNA Polymerase β (Polβ), we find that mutation of this surface threonine residue impacts critical Polβ protein-protein interactions. We show that proteasome-mediated degradation of Polβ is regulated by both ubiquitin-dependent and ubiquitin-independent processes via unique protein-protein interactions. The ubiquitin-independent proteasome pathway regulates the stability of Polβ in the cytosol via interaction between Polβ and NAD(P)H quinone dehydrogenase 1 (NQO1) in an NADH-dependent manner. Conversely, the interaction of Polβ with the scaffold protein X-ray repair cross complementing 1 (XRCC1) plays a role in the localization of Polβ to the nuclear compartment and regulates the stability of Polβ via a ubiquitin-dependent pathway. Further, we find that oxidative stress promotes the dissociation of the Polβ/NQO1 complex, enhancing the interaction of Polβ with XRCC1. Our results reveal that somatic mutations such as T304I in Polβ impact critical protein-protein interactions, altering the stability and sub-cellular localization of Polβ and providing mechanistic insight into how key protein-protein interactions regulate cellular responses to stress.

The effectiveness of many DNA-damaging chemotherapeutic drugs depends on their ability to form monoadducts, intrastrand crosslinks and/or interstrand crosslinks (ICLs) that interfere with transcription and replication. The ERCC1-XPF endonuclease plays a critical role in removal of these lesions by incising DNA either as part of nucleotide excision repair (NER) or interstrand crosslink repair (ICLR). Engagement of ERCC1-XPF in NER is well characterized and is facilitated by binding to the XPA protein. However, ERCC1-XPF recruitment to ICLs is less well understood. Moreover, specific mutations in XPF have been found to disrupt its function in ICLR but not in NER, but whether this involves differences in lesion targeting is unknown. Here, we imaged GFP-tagged ERCC1, XPF and ICLR-defective XPF mutants to investigate how in human cells ERCC1-XPF is localized to different types of psoralen-induced DNA lesions, repaired by either NER or ICLR. Our results confirm its dependence on XPA in NER and furthermore show that its engagement in ICLR is dependent on FANCD2. Interestingly, we find that two ICLR-defective XPF mutants (R689S and S786F) are less well recruited to ICLs. These studies highlight the differential mechanisms that regulate ERCC1-XPF activity in DNA repair.

Nucleotide Excision Repair (NER), which removes a variety of helix-distorting lesions from DNA, is initiated by two distinct DNA damage-sensing mechanisms. Transcription Coupled Repair (TCR) removes damage from the active strand of transcribed genes and depends on the SWI/SNF family protein CSB. Global Genome Repair (GGR) removes damage present elsewhere in the genome and depends on damage recognition by the XPC/RAD23/Centrin2 complex. Currently, it is not well understood to what extent both pathways contribute to genome maintenance and cell survival in a developing organism exposed to UV light. Here, we show that eukaryotic NER, initiated by two distinct subpathways, is well conserved in the nematode Caenorhabditis elegans. In C. elegans, involvement of TCR and GGR in the UV-induced DNA damage response changes during development. In germ cells and early embryos, we find that GGR is the major pathway contributing to normal development and survival after UV irradiation, whereas in later developmental stages TCR is predominantly engaged. Furthermore, we identify four ISWI/Cohesin and four SWI/SNF family chromatin remodeling factors that are implicated in the UV damage response in a developmental stage dependent manner. These in vivo studies strongly suggest that involvement of different repair pathways and chromatin remodeling proteins in UV-induced DNA repair depends on developmental stage of cells.

Global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) protect cells against a variety of helix-distorting DNA lesions. In C. elegans, GG-NER primarily acts in proliferative germ cells and embryos, while TC-NER acts in post-mitotic somatic cells to maintain transcription. We leverage this difference to distinguish whether proteins function in GG-NER and/or TC-NER by straightforward UV survival assays. Here, we detail a protocol for these assays, using GG-NER factor xpc-1 and TC-NER factor csb-1 as examples. For complete details on the use and execution of this protocol, please refer to Sabatella et al. (2021).

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.

DNA damage sensors DDB2 and XPC initiate global genome nucleotide excision repair (NER) to protect DNA from mutagenesis caused by helix-distorting lesions. XPC recognizes helical distortions by binding to unpaired ssDNA opposite DNA lesions. DDB2 binds to UV-induced lesions directly and facilitates efficient recognition by XPC. We show that not only lesion-binding but also timely DDB2 dissociation is required for DNA damage handover to XPC and swift progression of the multistep repair reaction. DNA-binding-induced DDB2 ubiquitylation and ensuing degradation regulate its homeostasis to prevent excessive lesion (re)binding. Additionally, damage handover from DDB2 to XPC coincides with the arrival of the TFIIH complex, which further promotes DDB2 dissociation and formation of a stable XPC-TFIIH damage verification complex. Our results reveal a reciprocal coordination between DNA damage recognition and verification within NER and illustrate that timely repair factor dissociation is vital for correct spatiotemporal control of a multistep repair process.

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we applied a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis showed that DDA1 is an integral component of the CRL4CSA complex. Functional analysis revealed that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.