XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair1,2. Here, we show that biallelic mutations in human XRCC1 are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. XRCC1-mutant patient cells exhibit not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation; a phenotype recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP3-5 and implicating 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.

A critical step of DNA single-strand break repair is the rapid recruitment of the scaffold protein XRCC1 that interacts with, stabilizes and stimulates multiple enzymatic components of the repair process. XRCC1 recruitment is promoted by PARP1, an enzyme that is activated following DNA damage and synthesizes ADP-ribose polymers that XRCC1 binds directly. However, cells possess two other DNA strand break-induced PARP enzymes, PARP2 and PARP3, for which the roles are unclear. To address their involvement in the recruitment of endogenous XRCC1 into oxidized chromatin we have established ‘isogenic’ human diploid cells in which PARP1 and/or PARP2, or PARP3 are deleted. Surprisingly, we show that either PARP1 or PARP2 are sufficient for near-normal XRCC1 recruitment at oxidative single-strand breaks (SSBs) as indicated by the requirement for loss of both proteins to greatly reduce or ablate XRCC1 chromatin binding following H2O2 treatment. Similar results were observed for PNKP; an XRCC1 protein partner important for repair of oxidative SSBs. Notably, concentrations of PARP inhibitor >1000-fold higher than the IC50 were required to ablate both ADP-ribosylation and XRCC1 chromatin binding following H2O2 treatment. These results demonstrate that very low levels of ADP-ribosylation, synthesized by either PARP1 or PARP2, are sufficient for XRCC1 recruitment following oxidative stress.

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

Summary Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes “trapped” on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1 −/− cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an “anti-trapper” that prevents toxic PARP1 activity.

12DNA topoisomerases are required to resolve DNA topological stress. Despite this essential 13 role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, 14 jeopardising genome stability. Here, to understand the genomic distribution and 15 mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA 16 cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human 17 genomes-and use the meiotic Spo11 protein to validate the broad applicability of this 18 method to explore the role of diverse topoisomerase family members. Our data 19 characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions 20 of Top2 activity in humans: tightly localised CTCF-proximal and broadly distributed 21 transcription-proximal. Moreover, the nucleotide resolution accuracy of our assay reveals 22 the influence primary DNA sequence has upon Top2 cleavage-for the first time 23 distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA 24 single-strand breaks (SSBs) induced by etoposide (VP16) in vivo. 25 26 27 101 1995; Keeney et al., 1997), an orthologue of archaeal Topoisomerase VI (Bergerat et al., 102 1997). We first verified this enrichment principle using meiotic sae2Δ cells, in which Spo11-103 linked DSBs are known to accumulate at defined loci due to abrogation of the nucleolytic 104 pathway that releases Spo11 (Keeney and Kleckner, 1995; Neale et al., 2005). To 105 demonstrate specific enrichment of protein-linked molecules, total genomic DNA from 106 Gittens et al. 2019 meiotic S. cerevisiae cells was isolated in the absence of proteolysis, digested with PstI 107 restriction enzyme, and isolated on glass-fibre spin columns (Figure 1A, Methods). We 108 used eluted material to assay a known Spo11-DSB hotspot by Southern blotting (Figure 109 1B). While DSB fragments are a minor fraction of input material (~10% of total), and were 110 absent in wash fractions, DSBs accounted for >99% of total eluted material, indicating 111 ~1000-fold enrichment relative to non-protein-linked DNA (Figure 1B). 112 113 CC-seq maps known Spo11-DSB hotspots genome-wide with high reproducibility 114 To generate a genome-wide map of Spo11-DSBs, genomic DNA from meiotic sae2∆ cells 115 was sonicated to <400 bp in length, enriched upon the silica column, eluted, and ligated to 116 DNA adapters in a two-step procedure that utilised the known phosphotyrosyl-unlinking 117 activity of mammalian TDP2 to uncap the Spo11-bound end ('CC-seq'; Figure 1A / 118 Methods) (Cortes Ledesma et al., 2009; Johnson et al., 2019). Libraries were paired-end 119 sequenced and mapped to the S. cerevisiae reference genome (Table S1) alongside reads 120 from a previous mapping technique ('Spo11-oligo-seq') that relies on the isolation of 121 Spo11-linked oligonucleotides generated in wild-type cells during DSB repair (Pan et al., 122 2011). CC-seq revealed sharp, localised peaks ('hotspots') in SPO11+ cells (Figure 1C, 123 middle) that visually (Figu...