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Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1 −/− mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1 −/− cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease. Adamowicz et al. report that toxic PARP1 activity, induced by ataxia-associated mutations in XRCC1, impairs the recovery of global transcription during DNA base excision repair by promoting aberrant recruitment and activity of the histone ubiquitin protease USP3.

Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3 -mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair. Defects in DNA single-strand break repair are associated with neurodegenerative disease. Here the authors reveal that mutations in ARH3 interfere with the catabolism of mono-(ADP-ribose) and lead to its accumulation on core histones following repair of endogenous or exogenous DNA single-strand breaks.

Mutations in TDP2, encoding tyrosyl-DNA phosphodiesterase 2, have been associated with a syndromal form of autosomal recessive spinocerebellar ataxia, type 23 (SCAR23). This is a very rare and progressive neurodegenerative disorder described in only nine patients to date, and caused by splice site or nonsense mutations that result in greatly reduced or absent TDP2 protein. TDP2 is required for the rapid repair of DNA double-strand breaks induced by abortive DNA topoisomerase II (TOP2) activity, important for genetic stability in post-mitotic cells such as neurons. Here, we describe a sibship that is homozygous for the first TDP2 missense mutation (p.Glu152Lys) and which presents with clinical features overlapping both SCAR23 and Fanconi anemia (FA). We show that in contrast to previously reported SCAR23 patients, fibroblasts derived from the current patient retain significant levels of TDP2 protein. However, this protein is catalytically inactive, resulting in reduced rates of repair of TOP2-induced DNA double-strand breaks and cellular hypersensitivity to the TOP2 poison, etoposide. The TDP2-mutated patient-derived fibroblasts do not display increased chromosome breakage following treatment with DNA crosslinking agents, but both TDP2-mutated and FA cells exhibit increased chromosome breakage in response to etoposide. This suggests that the FA pathway is required in response to TOP2-induced DNA lesions, providing a possible explanation for the clinical overlap between FA and the current TDP2-mutated patients. When reviewing the relatively small number of patients with SCAR23 that have been reported, it is clear that the phenotype of such patients can extend beyond neurological features, indicating that the TDP2 protein influences not only neural homeostasis but also other tissues as well.

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1.sup.-/- mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1.sup.-/- cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.

Background Microcephaly with early‐onset seizures (MCSZ) is a neurodevelopmental disorder caused by pathogenic variants in the DNA strand break repair protein, polynucleotide kinase 3′‐phosphatase (PNKP). Methods We have used whole genome sequencing and Sanger sequencing to identify disease‐causing variants, followed by a minigene assay, Western blotting, alkaline comet assay, γH2AX, and ADP‐ribose immunofluorescence. Results Here, we describe a patient with compound heterozygous variants in PNKP, including a missense variant in the DNA phosphatase domain (T323M) and a novel splice acceptor site variant within the DNA kinase domain that we show leads to exon skipping. We show that primary fibroblasts derived from the patient exhibit greatly reduced levels of PNKP protein and reduced rates of DNA single‐strand break repair, confirming that the mutated PNKP alleles are dysfunctional. Conclusion The data presented show that the detected compound heterozygous variants result in reduced levels of PNKP protein, which affect the repair of both oxidative and TOP1‐induced single‐strand breaks, and most likely causes MCSZ in this patient. Microcephaly with early‐onset seizures (MSCZ) is a neurodevelopmental disorder caused by pathogenic variants in the DNA strand break repair protein, polynucleotide kinase 3′‐phophatase (PNKP). Here, we describe a patient with compound heterozygous variants in PNKP, one missense variant, and a novel splice acceptor site variant which leads to exon skipping. We show that primary fibroblasts derived from the patient exhibit greatly reduced levels of PNKP protein and reduced rates of DNA single‐strand break repair, confirming that the mutated PNKP alleles are dysfunctional.