UVSSA facilitates transcription-coupled repair of DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) are covalent bonds between bases on opposing strands of the DNA helix which prevent DNA melting and subsequent DNA replication or RNA transcription. Here, we show that Ultraviolet Stimulated Scaffold Protein A (UVSSA) is critical for ICL repair in human cells, at least in part via the transcription coupled ICL repair (TC-ICR) pathway. Inactivation of UVSSA sensitizes human cells to ICL-inducing drugs, and delays ICL repair. UVSSA is required for replication-independent repair of a single ICL in a fluorescence-based reporter assay. UVSSA localizes to chromatin following ICL damage, and interacts with transcribing Pol II, CSA, CSB, and TFIIH. Specifically, UVSSA interaction with TFIIH is required for ICL repair and transcription inhibition blocks localization of transcription coupled repair factors to ICL damaged chromatin. Finally, UVSSA expression positively correlates with ICL-based chemotherapy resistance in human cancer cell lines. Our data strongly suggest that UVSSA is a novel ICL repair factor functioning in TC-ICR. These results provide further evidence that TC-ICR is a bona fide ICL repair mechanism that contributes to crosslinker drug resistance independently of replication-coupled ICL repair.

Comparing Mfd- and UvrD-dependent models of transcription coupled DNA repair in live Escherichia coli using single-molecule tracking.

During transcription-coupled DNA repair (TCR) the detection of DNA damage and initiation of nucleotide excision repair (NER) is performed by translocating RNA polymerases (RNAP), which are arrested upon encountering bulky DNA lesions. Two opposing models of the subsequent steps of TCR in bacteria exist. In the first model, stalled RNAPs are removed from the damage site by recruitment of Mfd which dislodges RNAP by pushing it forwards before recruitment of UvrA and UvrB. In the second model, UvrD helicase backtracks RNAP from the lesion site. Recent studies have proposed that both UvrD and UvrA continuously associate with RNAP before damage occurs, which forms the primary damage sensor for NER. To test these two models of TCR in living E. coli, we applied super-resolution microscopy (PALM) combined with single particle tracking to directly measure the mobility and recruitment of Mfd, UvrD, UvrA, and UvrB to DNA during ultraviolet-induced DNA damage. The intracellular mobilities of NER proteins in the absence of DNA damage showed that most UvrA molecules could in principle be complexed with RNAP, however, this was not the case for UvrD. Upon DNA damage, Mfd recruitment to DNA was independent of the presence of UvrA, in agreement with its role upstream of this protein in the TCR pathway. In contrast, UvrD recruitment to DNA was strongly dependent on the presence of UvrA. Inhibiting transcription with rifampicin abolished Mfd DNA-recruitment following DNA damage, whereas significant UvrD, UvrA, and UvrB recruitment remained, consistent with a UvrD and UvrA performing their NER functions independently of transcribing RNAP. Together, although we find that up to ∼8 UvrD-RNAP-UvrA complexes per cell could potentially form in the absence of DNA damage, our live-cell data is not consistent with this complex being the primary DNA damage sensor for NER.

Live-cell imaging of endogenous CSB-mScarletI as a sensitive marker for DNA-damage-induced transcription stress.

Transcription by RNA polymerase II (RNA Pol II) is crucial for cellular function, but DNA damage severely impedes this process. Thus far, transcription-blocking DNA lesions (TBLs) and their repair have been difficult to quantify in living cells. To overcome this, we generated, using CRISPR-Cas9-mediated gene editing, mScarletI-tagged Cockayne syndrome group B protein (CSB) and UV-stimulated scaffold protein A (UVSSA) knockin cells. These cells allowed us to study the binding dynamics of CSB and UVSSA to lesion-stalled RNA Pol II using fluorescence recovery after photobleaching (FRAP). We show that especially CSB mobility is a sensitive transcription stress marker at physiologically relevant DNA damage levels. Transcription-coupled nucleotide excision repair (TC-NER)-mediated repair can be assessed by studying CSB immobilization over time. Additionally, flow cytometry reveals the regulation of CSB protein levels by CRL4CSA-mediated ubiquitylation and deubiquitylation by USP7. This approach allows the sensitive detection of TBLs and their repair and the study of TC-NER complex assembly and stability in living cells.

Elf1 promotes Rad26's interaction with lesion-arrested Pol II for transcription-coupled repair.

Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a common mechanism to recognize a stalled Pol II, with additional interactions when Pol II is arrested at a lesion. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces further interactions between Rad26 and a lesion-arrested Pol II. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation. Together, our results provide important mechanistic insights into how two conserved transcription-coupled repair factors, Rad26/CSB and Elf1/ELOF1, work together at the initial lesion recognition steps of transcription-coupled repair.

Helicases required for nucleotide excision repair: structure, function and mechanism.

Nucleotide excision repair (NER) is a major DNA repair pathway conserved from bacteria to humans. Various DNA helicases, a group of enzymes capable of separating DNA duplex into two strands through ATP binding and hydrolysis, are required by NER to unwind the DNA duplex around the lesion to create a repair bubble and for damage verification and removal. In prokaryotes, UvrB helicase is required for repair bubble formation and damage verification, while UvrD helicase is responsible for the removal of the excised damage containing single-strand (ss) DNA fragment. In addition, UvrD facilitates transcription-coupled repair (TCR) by backtracking RNA polymerase stalled at the lesion. In eukaryotes, two helicases XPB and XPD from the transcription factor TFIIH complex fulfill the helicase requirements of NER. Interestingly, homologs of all these four helicases UvrB, UvrD, XPB, and XPD have been identified in archaea. This review summarizes our current understanding about the structure, function, and mechanism of these four helicases.

In vitro DNA repair genomics using XR-seq with Escherichia coli and mammalian cell-free extracts.

The XR-seq (eXcision Repair-sequencing) method has been extensively used to map nucleotide excision repair genome-wide in organisms ranging from Escherichia coli to yeast, Drosophila, Arabidopsis, mice, and humans. The basic feature of the method is to capture the excised oligomers carrying DNA damage, sequence them, and align their sequences to the genome. We wished to perform XR-seq in vitro with cell-free extract supplemented with a damaged DNA substrate so as to have greater flexibility in investigating factors that affect nucleotide excision repair in the cellular context [M. J. Smerdon, J. J. Wyrick, S. Delaney, J. Biol. Chem. 299, 105118 (2023)]. We report here the successful use of ultraviolet light-irradiated plasmids as substrates for repair in vitro and in vivo by E. coli and E. coli cell-free extracts and by mammalian cell-free extract. XR-seq analyses demonstrated common excision product length and sequence characteristics in vitro and in vivo for both the bacterial and mammalian systems. This approach is expected to help understand the effects of epigenetics and other cellular factors and conditions on DNA repair.

Transcription-coupled global genomic repair in E. coli.

The nucleotide excision repair (NER) pathway removes helix-distorting lesions from DNA in all organisms. Escherichia coli has long been a model for understanding NER, which is traditionally divided into major and minor subpathways known as global genome repair (GGR) and transcription-coupled repair (TCR), respectively. TCR has been assumed to be mediated exclusively by Mfd, a DNA translocase of minimal NER phenotype. This review summarizes the evidence that shaped the traditional view of NER in bacteria, and reviews data supporting a new model in which GGR and TCR are inseparable. In this new model, RNA polymerase serves both as the essential primary sensor of bulky DNA lesions genome-wide and as the delivery platform for the assembly of functional NER complexes in living cells.

Structural characterization of transcription-coupled repair protein UVSSA and its interaction with TFIIH protein.

UV-stimulated scaffold protein A (UVSSA) is a key protein in the Transcription-Coupled Nucleotide Excision Repair (TC-NER) pathway. UVSSA, an intrinsically disordered protein, interacts with multiple members of the pathway, tethering them into the complex. Several studies have reported that UVSSA recruits Transcription Factor IIH (TFIIH) via direct interaction, following which CSB is degraded and the lesion recognition TC-NER complex dissociates from the damage site to facilitate the DNA repair. Structural insights into these events remain largely unknown. Herein, we have investigated the interaction of human UVSSA with the Pleckstrin-Homology-domain of p62 subunit of TFIIH (p62-PHD) using biophysical techniques. We observed that UVSSA forms a stable complex with the p62-PHD in vitro. Small-angle scattering measurements using X-rays and neutrons revealed a significant change in pair-distance distribution function for UVSSA662/p62-PHD complex compared to UVSSA alone. Additionally, a significant decrease was observed in the radius of gyration of the complex. Our findings suggest that TFIIH binding to UVSSA causes significant conformational changes in UVSSA. We hypothesize that these conformational changes play an important role in the dissociation of the lesion recognition TC-NER complex.

New roles for elongation factors in RNA polymerase II ubiquitylation and degradation.

RNA polymerase II (RNAPII) encounters numerous impediments on its way to completing mRNA synthesis across a gene. Paused and arrested RNAPII are reactivated or rescued by elongation factors that travel with polymerase as it transcribes DNA. However, when RNAPII fails to resume transcription, such as when it encounters an unrepairable bulky DNA lesion, it is removed by the targeting of its largest subunit, Rpb1, for degradation by the ubiquitin-proteasome system (UPS). We are starting to understand this process better and how the UPS marks Rbp1 for degradation. This review will focus on the latest developments and describe new functions for elongation factors that were once thought to only promote elongation in unstressed conditions in the removal and degradation of RNAPII. I propose that in addition to changes in RNAPII structure, the composition and modification of elongation factors in the elongation complex determine whether to rescue or degrade RNAPII.

Oxidative stress drives mutagenesis through transcription-coupled repair in bacteria.

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one of the main public health problems of the twenty-first century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics and highly divergent species tested, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies a highly conserved pathway that drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.

Structural Basis of Damaged Nucleotide Recognition by Transcribing RNA Polymerase II in the Nucleosome.

In transcription-coupled repair (TCR), transcribing RNA polymerase II (RNAPII) stalls at a DNA lesion and recruits TCR proteins to the damaged site. However, the mechanism by which RNAPII recognizes a DNA lesion in the nucleosome remains enigmatic. In the present study, we inserted an apurinic/apyrimidinic DNA lesion analogue, tetrahydrofuran (THF), in the nucleosomal DNA, where RNAPII stalls at the SHL(-4), SHL(-3.5), and SHL(-3) positions, and determined the structures of these complexes by cryo-electron microscopy. In the RNAPII-nucleosome complex stalled at SHL(-3.5), the nucleosome orientation relative to RNAPII is quite different from those in the SHL(-4) and SHL(-3) complexes, which have nucleosome orientations similar to naturally paused RNAPII-nucleosome complexes. Furthermore, we found that an essential TCR protein, Rad26 (CSB), enhances the RNAPII processivity, and consequently augments the DNA damage recognition efficiency of RNAPII in the nucleosome. The cryo-EM structure of the Rad26-RNAPII-nucleosome complex revealed that Rad26 binds to the stalled RNAPII through a novel interface, which is completely different from those previously reported. These structures may provide important information to understand the mechanism by which RNAPII recognizes the nucleosomal DNA lesion and recruits TCR proteins to the stalled RNAPII on the nucleosome.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

The Mfd protein is the transcription-repair coupling factor (TRCF) in Mycobacterium smegmatis.

In vitro and in vivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E. coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

Transcription coupled DNA repair protein UVSSA binds to DNA and RNA: Mapping of nucleic acid interaction sites on human UVSSA.

Transcription-coupled repair (TCR) is a dedicated pathway for the preferential repair of bulky transcription-blocking DNA lesions. These lesions stall the elongating RNA-polymerase II (RNAPII) triggering the recruitment of TCR proteins at the damaged site. UV-stimulated scaffold protein A (UVSSA) is a recently identified cofactor which is involved in stabilization of the TCR complex, recruitment of DNA-repair machinery and removal/restoration of RNAPII from the lesion site. Mutations in UVSSA render the cells TCR-deficient and have been linked to UV-sensitive syndrome. Human UVSSA is a 709-residue long protein with two short conserved domains; an N-terminal (residues 1-150) and a C-terminal (residues 495-605) domain, while the rest of the protein is predicted to be intrinsically disordered. The protein is well conserved in eukaryotes, however; none of its homologs have been characterized yet. Here, we have purified the recombinant human UVSSA and have characterized it using bioinformatics, biophysical and biochemical techniques. Using EMSA, SPR and fluorescence-based methods, we have shown that human UVSSA interacts with DNA and RNA. Furthermore, we have mapped the nucleic acid binding regions using several recombinant protein fragments containing either the N-terminal or the C-terminal domains. Our data indicate that UVSSA possesses at least two nucleic acid binding regions; the N-terminal domain and a C-terminal tail region (residues 606-662). These regions, far apart in sequence space, are predicted to be in close proximity in structure-space suggesting a coherent interaction with target DNA/RNA. The study may provide functional clues about the novel family of UVSSA proteins.

Aicardi-Goutières syndrome with SAMHD1 deficiency can be diagnosed by unscheduled DNA synthesis test.

Aicardi-Goutières syndrome (AGS) is a rare genetic disorder characterised by progressive encephalopathy, involving microcephaly, intracranial calcification, and cerebrospinal fluid lymphocytosis with increased interferon-α concentrations. The clinical features of AGS overlap with fetal cerebral anomalies caused by congenital infections, such as TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes), or with those of other genetic disorders showing neonatal microcephaly, including Cockayne syndrome (CS) with transcription-coupled DNA repair deficiency, and Seckel syndrome (SS) showing aberrant cell-cycle checkpoint signaling. Therefore, a differential diagnosis to confirm the genetic cause or a proof of infection should be considered. In this report, we describe an individual who showed primordial dwarfism and encephalopathy, and whose initial diagnosis was CS. First, we conducted conventional DNA repair proficiency tests for the patient derived fibroblast cells. Transcription-coupled nucleotide excision repair (TC-NER) activity, which is mostly compromised in CS cases, was slightly reduced in the patient's cells. However, unscheduled DNA synthesis (UDS) was significantly diminished. These cellular traits were inconsistent with the diagnosis of CS. We further performed whole exome sequencing for the case and identified a compound heterozygous loss-of-function variants in the SAMHD1 gene, mutations in which are known to cause AGS. As SAMHD1 encodes deoxyribonucleoside triphosphate triphosphohydrolase, we reasoned that the deoxyribonucleoside triphosphate (dNTP) pool size in the patient's cells was elevated, and the labeling efficiency of UDS-test was hindered due to the reduced concentration of phosphorylated ethynyl deoxyuridine (EdU), a nucleoside analogue used for the assay. In conclusion, UDS assay may be a useful diagnostic tool to distinguish between AGS with SAMHD1 mutations and other related diseases.

A matter of delicate balance: Loss and gain of Cockayne syndrome proteins in premature aging and cancer.

DNA repair genes are critical for preserving genomic stability and it is well established that mutations in DNA repair genes give rise to progeroid diseases due to perturbations in different DNA metabolic activities. Cockayne Syndrome (CS) is an autosomal recessive inheritance caused by inactivating mutations in CSA and CSB genes. This review will primarily focus on the two Cockayne Syndrome proteins, CSA and CSB, primarily known to be involved in Transcription Coupled Repair (TCR). Curiously, dysregulated expression of CS proteins has been shown to exhibit differential health outcomes: lack of CS proteins due to gene mutations invariably leads to complex premature aging phenotypes, while excess of CS proteins is associated with carcinogenesis. Thus it appears that CS genes act as a double-edged sword whose loss or gain of expression leads to premature aging and cancer. Future mechanistic studies on cell and animal models of CS can lead to potential biological targets for interventions in both aging and cancer development processes. Some of these exciting possibilities will be discussed in this review in light of the current literature.

Nucleotide excision repair: a versatile and smart toolkit.

Nucleotide excision repair (NER) is a major pathway to deal with bulky adducts induced by various environmental toxins in all cellular organisms. The two sub-pathways of NER, global genome repair (GGR) and transcription-coupled repair (TCR), differ in the damage recognition modes. In this review, we describe the molecular mechanism of NER in mammalian cells, especially the details of damage recognition steps in both sub-pathways. We also introduce new sequencing methods for genome-wide mapping of NER, as well as recent advances about NER in chromatin by these methods. Finally, the roles of NER factors in repairing oxidative damages and resolving R-loops are discussed.

Transcription and genome integrity.

Transcription can cause genome instability by promoting R-loop formation but also act as a mutation-suppressing machinery by sensing of DNA lesions leading to the activation of DNA damage signaling and transcription-coupled repair. Recovery of RNA synthesis following the resolution of repair of transcription-blocking lesions is critical to avoid apoptosis and several new factors involved in this process have recently been identified. Some DNA repair proteins are recruited to initiating RNA polymerases and this may expediate the recruitment of other factors that participate in the repair of transcription-blocking DNA lesions. Recent studies have shown that transcription of protein-coding genes does not always give rise to spliced transcripts, opening the possibility that cells may use the transcription machinery in a splicing-uncoupled manner for other purposes including surveillance of the transcribed genome.

Suppressor mutations in Escherichia coli RNA polymerase alter transcription initiation but do not affect translesion RNA synthesis in vitro.

Bacterial RNA polymerase (RNAP) coordinates transcription with DNA repair and replication. Many RNAP mutations have pleiotropic phenotypes with profound effects on transcription-coupled processes. One class of RNAP mutations (rpo∗) has been shown to suppress mutations in regulatory factors responsible for changes in gene expression during stationary phase or starvation, as well as in factors involved in the restoration of replication forks after DNA damage. These mutations were suggested to affect the ability of RNAP to transcribe damaged DNA and to decrease the stability of transcription complexes, thus facilitating their dislodging during DNA replication and repair, although this was not explicitly demonstrated. Here, we obtained nine mutations of this class located around the DNA/RNA binding cleft of Escherichia coli RNAP and analyzed their transcription properties in vitro. We found that these mutations decreased promoter complex stability to varying degrees, and all decreased the activity of rRNA promoters. However, they did not have strong effects on elongation complex stability. Some mutations were shown to stimulate transcriptional pauses or decrease intrinsic RNA cleavage by RNAP, but none altered the ability of RNAP to transcribe DNA templates containing damaged nucleotides. Thus, we conclude that the suppressor phenotypes of the mutations are unlikely to result from direct effects on DNA lesion recognition by RNAP but may be primarily explained by changes in transcription initiation. Further analysis of the effects of these mutations on the genomic distribution of RNAP and its interactions with regulatory factors will be essential for understanding their diverse phenotypes in vivo.

Transcription-coupled DNA repair underlies variation in persister awakening and the emergence of resistance.

Persisters constitute a population of temporarily antibiotic-tolerant variants in an isogenic bacterial population and are considered an important cause of relapsing infections. It is currently unclear how cellular damage inflicted by antibiotic action is reversed upon persister state exit and how this relates to antibiotic resistance development at the molecular level. We demonstrate that persisters, upon fluoroquinolone treatment, accumulate oxidative DNA damage, which is repaired through nucleotide excision repair. Detection of the damage occurs via transcription-coupled repair using UvrD-mediated backtracking or Mfd-controlled displacement of the RNA polymerase. This competition results in heterogeneity in persister awakening lags. Most persisters repair the oxidative DNA damage, displaying a mutation rate equal to the untreated population. However, the promutagenic factor Mfd increases the mutation rate in a persister subpopulation. Our data provide in-depth insight into the molecular mechanisms underlying persister survival and pinpoint Mfd as an important molecular factor linking persistence to resistance development.