PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf(-/-) B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions.

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.

Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/ DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.

Here, we identify such an enzyme in human cells and show that this activity efficiently restores 5'-phosphate termini at DNA double-strand breaks in preparation for DNA ligation. We reveal that this enzyme is TTRAP, a member of the Mg 2+ /Mn 2+ -dependent family of phosphodiesterases, and show that cellular depletion of TTRAP results in increased susceptibility and sensitivity to topoisomerase II-induced DNA double-strand breaks. TTRAP is the first human 5'-tyrosine phosphodiesterase to be identified and we suggest this enzyme additionally be denoted tyrosyl DNA phosphodiesterase-2 (TDP2).In an attempt to identify novel human tyrosyl DNA phosphodiesterase activities, we exploited the hypersensitivity of Saccharomyces cerevisiae tdp1Δ rad1Δ double mutant cells to camptothecin (CPT), a topoisomerase I (Top1) poison that induces single-strand breaks (SSBs) with Top1 covalently linked to the 3'-terminus 3, 10 . This strain lacks not only Tdp1 but also Rad1-Rad10 nuclease, which in yeast provides an alternative (endonucleolytic) pathway for removing Top1 from 3'-termini 11,12 . We transformed this strain with a human cDNA library 3 and screened the resulting population of transformants for cellular resistance to CPT. Of six tdp1Δ rad1Δ transformants displaying wild-type levels of CPT resistance, three harboured cDNA clones encoding TDP1 and three harboured cDNA clones encoding TTRAP (TRAF and TNF receptor-associated protein), a protein of unknown function and a putative member of the Mg 2+ /Mn 2+ -dependent phosphodiesterase super-family, with the DNA repair protein AP endonuclease-1 (APE1) being its closest relative 13,14 (Fig.1a and Supplementary Fig.1).The TDP1 and TTRAP cDNA clones recovered in the genetic screen suppressed the CPT sensitivity of tdp1Δ rad1Δ cells to a similar extent (Fig. 1b & data not shown). Whilst the pACT-TTRAP clones encoded TTRAP protein that lacked eight (pACT-TTRAP-2, pACT-TTRAP-3) or twenty-two (pACT-TTRAP-1) residues from the amino-terminus (data not shown), full-length TTRAP similarly suppressed the CPT sensitivity of tdp1Δ rad1Δ (Fig. 1c, left). In contrast, human APE1 protein failed to suppress this sensitivity, suggesting that ability to complement CPT sensitivity in tdp1Δ rad1Δ cells is not a generic feature of metaldependent phosphodiesterases (Fig. 1c, left). Conversely, whereas human APE1 suppressed the sensitivity of AP endonuclease-defective apn1Δapn2Δ tpp1Δ yeast cells to methyl methanesulphonate (MMS)-induced DNA base damage, human TTRAP did not, suggesting that the impact of TTRAP in these experiments was restricted to topoisomerase-mediated DNA damage ( Fig. 1c, right). TTRAP contains four highly conserved motifs that putatively assign this protein to the metal-dependent phosphodiesterase superfamily (see Fig.1a and Supplementary Fig.1). We thus examined whether mutation of two predicted catalytic residues (Fig.1a; E152 and D262) within two of these motifs impacted on the complementation of CPT sensitivity by TTRAP. Indeed, in contrast to wild-type TTRAP protein, ne...

Amyotrophic lateral sclerosis (ALS) is associated with progressive degeneration of motor neurons. Several of the genes associated with this disease encode proteins involved in RNA processing, including fused-in-sarcoma/translocated-in-sarcoma (FUS/TLS). FUS is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind thousands of pre-mRNAs and can regulate their splicing. Here, we have examined the possibility that FUS is also a component of the cellular response to DNA damage. We show that both GFP-tagged and endogenous FUS re-localize to sites of oxidative DNA damage induced by UVA laser, and that FUS recruitment is greatly reduced or ablated by an inhibitor of poly (ADP-ribose) polymerase activity. Consistent with this, we show that recombinant FUS binds directly to poly (ADP-ribose) in vitro, and that both GFP-tagged and endogenous FUS fail to accumulate at sites of UVA laser induced damage in cells lacking poly (ADP-ribose) polymerase-1. Finally, we show that GFP-FUSR521G, harbouring a mutation that is associated with ALS, exhibits reduced ability to accumulate at sites of UVA laser-induced DNA damage. Together, these data suggest that FUS is a component of the cellular response to DNA damage, and that defects in this response may contribute to ALS.