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Cockayne syndrome B (CSB) is a multifaceted protein with known functions in DNA repair and transcription elongation. We recently demonstrated that CSB can recognize DNA secondary structures known as multimolecular G4s (mG4s) with high selectivity, but the potential biological significance of this interaction remains to be elucidated. In this study, we report that CSB can undergo liquid-liquid phase separation (LLPS), a process heavily linked with transcriptional regulation. Importantly, we found that the binding of CSB to mG4s promotes LLPS, leading to the physical segregation of DNA sequences containing mG4s within CSB-mG4 droplets from those lacking this structural motif. Furthermore, we revealed that mG4-binding alters the physicochemical properties of the phase-separated CSB, including increased salt resistance and decreased in-droplet mobility. Given the growing evidence supporting an active role for G4s in stimulating transcription, we anticipate that the selective LLPS displayed by CSB upon mG4 binding may be relevant in the context of transcriptional regulation.

Transcription-coupled nucleotide excision repair (TC-NER) safeguards transcription by repairing transcription-blocking lesions (TBLs), which are highly cytotoxic if unresolved. TC-NER is triggered when RNA Polymerase II (Pol II) stalls at a TBL and is recognized by CSB, followed by recruitment of the TC-NER factors CSA and UVSSA. Loss of any of these factors causes complete TC-NER deficiency. Mutations in CSB or CSA typically result in Cockayne syndrome (CS), a disorder marked by neurodegeneration and severe premature aging, highlighting the importance of TC-NER. By contrast, mutations in UVSSA cause the much milder UV-sensitive syndrome (UVSS), limited to cutaneous symptoms. Recently, this difference has been linked to the ability of UVSSA-deficient, but not CSB- or CSA-deficient, cells to clear stalled Pol II through proteasomal degradation, allowing alternative repair routes. Unexpectedly, patients with an early nonsense mutation in CSB (R77X), leading to undetectable protein levels, develop UVSS rather than CS. We examined this paradox and found that R77X cells can still degrade lesion-stalled Pol II, which is caused by residual but functional CSB expression in these cells. Together, our findings refine the model that defective Pol II processing is central to CS pathogenesis and extend its relevance across TC-NER related mutations.

Cockayne Syndrome type B (CSB) is caused by mutations in the ERCC6 gene, which encodes a key protein involved in transcription-coupled nucleotide excision repair (TC-NER) and chromatin remodeling. Deficiency in CSB leads to defective transcriptional recovery after DNA damage, oxidative stress accumulation, and progressive neurodegeneration. In this work, we generated a CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC) line, IUFi004-A-12, carrying a homozygous mutation in ERCC6 causing a premature stop codon in its 10th exon. The modified iPSCs displayed normal morphology, expressed pluripotency markers, and differentiated into all three germ layers. This model enables mechanistic studies of CSB dysfunction and facilitates therapeutic development for Cockayne Syndrome.

Rad26, a yeast homologue of mammalian Cockayne syndrome protein B (CSB), plays an essential role in transcription-coupled nucleotide excision repair (TC-NER). Rad26/CSB binds RNA polymerase II stalled at DNA lesions and recruits DNA repair factors, functioning as a molecular scaffold. In addition, Rad26/CSB possesses nucleosome-remodeling activity that may help restore transcription after DNA repair. Here we determine the cryo-electron microscopy structure of the Rad26/CSB-nucleosome complex. Rad26/CSB binds near the nucleosomal entry/exit region (superhelical location ±6) through a unique mechanism in which its ATPase domains, Lobe 1 and Lobe 2, engage nucleosomal DNA in a reverse orientation compared with other remodelers such as Snf2 and Ino80. Mutational, biochemical, and crosslinking mass-spectrometric analyses demonstrate the requirement of the KR loop for nucleosome binding and remodeling. Furthermore, we show that N-terminal auto-inhibition involves long-range contacts between the disordered N-terminus and the Lobe 2 region, and is relieved by mutations of Leu8 and Leu11. These findings reveal the structural basis of Rad26/CSB-mediated nucleosome remodeling in TC-NER.

BACKGROUND: Cockayne syndrome (CS), primarily caused by autosomal recessive pathogenic variants in ERCC6 (CSB) or ERCC8 (CSA), is a transcription-coupled nucleotide excision repair disorder. CS frequently presents with features similar to primary mitochondrial disease (PMD), including leukodystrophy, lactic acidemia, and skeletal muscle mitochondrial DNA (mtDNA) depletion. How this mitochondrial phenotype arises at the cellular level, and whether it can be pharmacologically targeted, is not yet clear. METHODS: We characterized mtDNA content, respiratory chain (RC) protein abundance, mitochondrial biogenesis signaling pathways, and oxidative phosphorylation capacity in primary fibroblasts from two siblings with identical compound heterozygous ERCC6 pathogenic variants (c.1526+1G>T; c.2800C>A, p.Pro934Thr) despite marked intrafamilial phenotypic divergence. A combined metabolic stress exposure (galactose, reduced glutamine, and buthionine sulfoximine, (BSO)) which reduced CS cell survival was used to screen for therapeutic leads among twenty-three candidate mitochondrial disease therapeutic compounds. Lead compounds were mechanistically validated at the level of mitochondrial superoxide, total cellular oxidative stress, glutathione, and autophagic flux. RESULTS: Patient fibroblasts exhibited several hallmarks of PMD, including reduced mtDNA content, decreased expression of complex I subunit NDUFB8, elevated expression of TOM20 with paradoxically decreased PGC1α suggestive of impaired mitophagic clearance, and decreased mitochondrial respiratory capacity. Under combined metabolic stress, ATP-levels indicative of survival in CS patient fibroblasts selectively collapsed to ~20% of controls. Five dual-rescue compounds, defined as agents that reproducibly restored ATP-based cell survival in both patient fibroblast lines under stress, were identified, including N-acetylcysteine (NAC), coenzyme Q10 (CoQ10), rapamycin, taurine, and (-)-epicatechin. Mechanistic profiling resolved three functional classes of therapeutic effects in CS cells: (1) upstream mitochondrial reactive oxygen species reduction (NAC, CoQ10); (2) mTORC1 inhibition bypassing defective stress-induced autophagic induction (rapamycin); and (3) extra-mitochondrial improvement in cellular stress resilience ((-)- epicatechin, taurine). CONCLUSIONS: ERCC6-based CSB deficiency produced a stress-sensitive and physiologically complex mitochondrial phenotype in patient fibroblasts that was pharmacologically treatable by targeting three mechanistically distinct pathways. Oxidative and broader stress buffering, autophagy modulation via mTORC1 inhibition, and enhanced cellular resilience highlight novel therapeutic opportunities to be advanced to clinical trials in CSB patients.