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Covalent DNA–protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA–protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS. Three studies identify a transcription-coupled DNA–protein cross-link repair pathway that depends on the Cockayne syndrome proteins and the proteasome.

Biallelic mutations in human XRCC1 are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cerebellar ataxia link to mutant XRCC1 This paper shows that mutated forms of human XRCC1, a scaffold protein involved in DNA single-strand break repair, are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. In cells from a patient with an XRCC1 −/− mutation, rates of break repair are reduced and the single-strand break sensor protein PARP1 is hyperactivated, resulting in abnormally high levels of cellular ADP-ribose. Genetic deletion of Parp1 in Xrcc1 -defective mice prevents the accumulation of excessive ADP-ribose and rescues the loss of cerebellar neurons and cerebellar ataxia. These findings point to PARP1 as a possible therapeutic target in DNA strand break repair-defective disease. XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair 1 , 2 . Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP 3 , 4 , 5 and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1 -defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease.

Temozolomide (TMZ) was used for the treatment of glioblastoma (GBM) for over a decade, but its treatment benefits are limited by acquired resistance, a process that remains incompletely understood. Here we report that an enhancer, located between the promoters of marker of proliferation Ki67 ( MKI67 ) and O6-methylguanine-DNA-methyltransferase ( MGMT ) genes, is activated in TMZ-resistant patient-derived xenograft (PDX) lines and recurrent tumor samples. Activation of the enhancer correlates with increased MGMT expression, a major known mechanism for TMZ resistance. We show that forced activation of the enhancer in cell lines with low MGMT expression results in elevated MGMT expression. Deletion of this enhancer in cell lines with high MGMT expression leads to a dramatic reduction of MGMT and a lesser extent of Ki67 expression, increased TMZ sensitivity, and impaired proliferation. Together, these studies uncover a mechanism that regulates MGMT expression, confers TMZ resistance, and potentially regulates tumor proliferation. Temozolomide (TMZ) resistance in glioblastomas (GBM) is associated with increased MGMT expression. Here, the authors identify an enhancer between the promoters of MKI67 and MGMT , that when activated drives MGMT expression despite MGMT promoter methylation to confer TMZ resistance in GBM.

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.

Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein–protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid–liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.

Actively transcribed regions of the genome are protected by transcription-coupled DNA repair mechanisms, including transcription-coupled homologous recombination (TC-HR). Here we used reactive oxygen species (ROS) to induce and characterize TC-HR at a transcribed locus in human cells. As canonical HR, TC-HR requires RAD51. However, the localization of RAD51 to damage sites during TC-HR does not require BRCA1 and BRCA2, but relies on RAD52 and Cockayne Syndrome Protein B (CSB). During TC-HR, RAD52 is recruited by CSB through an acidic domain. CSB in turn is recruited by R loops, which are strongly induced by ROS in transcribed regions. Notably, CSB displays a strong affinity for DNA:RNA hybrids in vitro, suggesting that it is a sensor of ROS-induced R loops. Thus, TC-HR is triggered by R loops, initiated by CSB, and carried out by the CSB-RAD52-RAD51 axis, establishing a BRCA1/2-independent alternative HR pathway protecting the transcribed genome. Transcription-coupled homologous recombination (TC-HR) is activated by reactive oxygen species-induced DNA damage to maintain transcribed genome stability. The authors demonstrate that R loops are induced by ROS at the transcribed genome, triggering a CSB-RAD52- dependent but BRCA1/2-independent RAD51 loading for repair.

Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug–genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.Two side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.

Tomoo Ogi and colleagues report mutations of UVSSA causing a third complementation group of the UV-sensitive syndrome. UVSSA deficiency results in defective transcription-coupled nucleotide-excision repair and failure to resolve stalled RNA polymerase IIo at DNA damage sites. UV-sensitive syndrome (UV S S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma 1 , 2 , 3 , 4 . Despite mild clinical features, cells from individuals with UV S S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER) 2 , 4 , 5 , which removes DNA damage in actively transcribed genes 6 . Three of the seven known UV S S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB , respectively) 7 , 8 . The remaining four individuals with UV S S, one of whom is described for the first time here, formed a separate UV S S-A complementation group 1 , 9 , 10 ; however, the responsible gene was unknown. Using exome sequencing 11 , we determine that mutations in the UVSSA gene (formerly known as KIAA1530 ) cause UV S S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders.

One of the most central questions about the repair of a double-strand DNA break (DSB) concerns how the two free DNA ends are brought together — a step called synapsis. Using single-molecule FRET (smFRET), we show here that both Ku plus XRCC4:DNA ligase IV are necessary and sufficient to achieve a flexible synapsis of blunt DNA ends, whereas either alone is not. Addition of XLF causes a transition to a close synaptic state, and maximum efficiency of close synapsis is achieved within 20 min. The promotion of close synapsis by XLF indicates a role that is independent of a filament structure, with action focused at the very ends of each duplex. DNA-PKcs is not required for the formation of either the flexible or close synaptic states. This model explains in biochemical terms the evolutionarily central synaptic role of Ku, X4L4, and XLF in NHEJ for all eukaryotes. During a process termed synapsis, the two DNA ends at a double-strand break (DSB) are brought together into physical proximity. Here, the authors use a single-molecule FRET approach with purified proteins to investigate the mechanism of synapsis in DSB repair by non-homologous DNA end joining (NHEJ).

Kathryn Lunetta and colleagues report a meta-analysis of 22 genome-wide association studies for age at menopause. They identify 13 loci newly associated with age at natural menopause, including several candidate genes with roles in DNA repair and immune function. To newly identify loci for age at natural menopause, we carried out a meta-analysis of 22 genome-wide association studies (GWAS) in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 loci newly associated with age at natural menopause (at P < 5 × 10 −8 ). Candidate genes located at these newly associated loci include genes implicated in DNA repair ( EXO1 , HELQ , UIMC1 , FAM175A , FANCI , TLK1 , POLG and PRIM1 ) and immune function ( IL11 , NLRP11 and PRRC2A (also known as BAT2 )). Gene-set enrichment pathway analyses using the full GWAS data set identified exoDNase, NF-κB signaling and mitochondrial dysfunction as biological processes related to timing of menopause.