Parp1 hyperactivity couples DNA breaks to aberrant neuronal calcium signalling and lethal seizures.

Defects in DNA single-strand break repair (SSBR) are linked with neurological dysfunction but the underlying mechanisms remain poorly understood. Here, we show that hyperactivity of the DNA strand break sensor protein Parp1 in mice in which the central SSBR protein Xrcc1 is conditionally deleted (Xrcc1Nes-Cre ) results in lethal seizures and shortened lifespan. Using electrophysiological recording and synaptic imaging approaches, we demonstrate that aberrant Parp1 activation triggers seizure-like activity in Xrcc1-defective hippocampus ex vivo and deregulated presynaptic calcium signalling in isolated hippocampal neurons in vitro. Moreover, we show that these defects are prevented by Parp1 inhibition or deletion and, in the case of Parp1 deletion, that the lifespan of Xrcc1Nes-Cre mice is greatly extended. This is the first demonstration that lethal seizures can be triggered by aberrant Parp1 activity at unrepaired SSBs, highlighting PARP inhibition as a possible therapeutic approach in hereditary neurological disease.

XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia.

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair. 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 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.

The Future of Precision Oncology.

Our understanding of the molecular mechanisms underlying cancer development and evolution have evolved rapidly over recent years, and the variation from one patient to another is now widely recognized. Consequently, one-size-fits-all approaches to the treatment of cancer have been superseded by precision medicines that target specific disease characteristics, promising maximum clinical efficacy, minimal safety concerns, and reduced economic burden. While precision oncology has been very successful in the treatment of some tumors with specific characteristics, a large number of patients do not yet have access to precision medicines for their disease. The success of next-generation precision oncology depends on the discovery of new actionable disease characteristics, rapid, accurate, and comprehensive diagnosis of complex phenotypes within each patient, novel clinical trial designs with improved response rates, and worldwide access to novel targeted anticancer therapies for all patients. This review outlines some of the current technological trends, and highlights some of the complex multidisciplinary efforts that are underway to ensure that many more patients with cancer will be able to benefit from precision oncology in the near future.

PARP-3 and APLF function together to accelerate nonhomologous end-joining.

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.

Non-homologous end joining: Common interaction sites and exchange of multiple factors in the DNA repair process.

Non-homologous end-joining (NHEJ) is the dominant means of repairing chromosomal DNA double strand breaks (DSBs), and is essential in human cells. Fifteen or more proteins can be involved in the detection, signalling, synapsis, end-processing and ligation events required to repair a DSB, and must be assembled in the confined space around the DNA ends. We review here a number of interaction points between the core NHEJ components (Ku70, Ku80, DNA-PKcs, XRCC4 and Ligase IV) and accessory factors such as kinases, phosphatases, polymerases and structural proteins. Conserved protein-protein interaction sites such as Ku-binding motifs (KBMs), XLF-like motifs (XLMs), FHA and BRCT domains illustrate that different proteins compete for the same binding sites on the core machinery, and must be spatially and temporally regulated. We discuss how post-translational modifications such as phosphorylation, ADP-ribosylation and ubiquitinylation may regulate sequential steps in the NHEJ pathway or control repair at different types of DNA breaks.

APLF promotes the assembly and activity of non-homologous end joining protein complexes.

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.

The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.

Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function.

PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage.

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-FUS(R521G), 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.

Identification and Functional Characterizations of N-Terminal α-N-Methylation and Phosphorylation of Serine 461 in Human Poly(ADP-ribose) Polymerase 3.

Poly(ADP-ribose) polymerase 3 (PARP3) is a member of the PARP family enzymes which catalyze the ADP-ribosylation of proteins. PARP3 plays an important role in DNA damage repair and mitotic progression. In this study, we identified, using mass spectrometric techniques, two novel post-translational modification sites in PARP3, α-N-methylation and phosphorylation of serine 461 (S461). We found that the N-terminal α-amino group of PARP3 is heavily methylated in human cells, and N-terminal RCC1 methyltransferase (NRMT) is a key enzyme required for this methylation. We also observed that the phosphorylation level of S461 in PARP3 could be reduced in human cells upon treatment with flavopiridol, a cyclin-dependent kinase inhibitor. Moreover, we demonstrated that S461 phosphorylation, but not α-N-methylation of PARP3, may be involved in the cellular response toward DNA double-strand breaks. These findings provide novel insights into the post-translational regulation of PARP3.

APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks.

Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H(2)O(2) or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells.

Acylpeptide hydrolase is a component of the cellular response to DNA damage.

Acylpeptide hydrolase (APEH) deacetylates N-alpha-acetylated peptides and selectively degrades oxidised proteins, but the biochemical pathways that are regulated by this protease are unknown. Here, we identify APEH as a component of the cellular response to DNA damage. Although APEH is primarily localised in the cytoplasm, we show that a sub-fraction of this enzyme is sequestered at sites of nuclear damage following UVA irradiation or following oxidative stress. We show that localization of APEH at sites of nuclear damage is mediated by direct interaction with XRCC1, a scaffold protein that accelerates the repair of DNA single-strand breaks. We show that APEH interacts with the amino-terminal domain of XRCC1, and that APEH facilitates both single-strand break repair and cell survival following exposure to H2O2 in human cells. These data identify APEH as a novel proteolytic component of the DNA damage response.

Ethanol modifies the effect of handling stress on gene expression: problems in the analysis of two-way gene expression studies in mouse brain.

Studies analysing the effects of acute treatments on animal behaviour and brain biochemistry frequently use pairwise comparisons between sham-treated and -untreated animals. In this study, we analyse expression of tPA, Grik2, Smarca2 and the transcription factor, Sp1, in mouse cerebellum following acute ethanol treatment. Expression is compared to saline-injected and -untreated control animals. We demonstrate that acute i.p. injection of saline may alter gene expression in a gene-specific manner and that ethanol may modify the effects of sham treatment on gene expression, as well as inducing specific effects independent of any handling related stress. In addition to demonstrating the complexity of gene expression in response to physical and environmental stress, this work raises questions on the interpretation and validity of studies relying on pairwise comparisons.

The Ku-binding motif is a conserved module for recruitment and stimulation of non-homologous end-joining proteins.

The Ku-binding motif (KBM) is a short peptide module first identified in APLF that we now show is also present in Werner syndrome protein (WRN) and in Modulator of retrovirus infection homologue (MRI). We also identify a related but functionally distinct motif in XLF, WRN, MRI and PAXX, which we denote the XLF-like motif. We show that WRN possesses two KBMs; one at the N terminus next to the exonuclease domain and one at the C terminus next to an XLF-like motif. We reveal that the WRN C-terminal KBM and XLF-like motif function cooperatively to bind Ku complexes and that the N-terminal KBM mediates Ku-dependent stimulation of WRN exonuclease activity. We also show that WRN accelerates DSB repair by a mechanism requiring both KBMs, demonstrating the importance of WRN interaction with Ku. These data define a conserved family of KBMs that function as molecular tethers to recruit and/or stimulate enzymes during NHEJ.

PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B(Glu2).

PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.

The PARP Inhibitor AZD2461 Provides Insights into the Role of PARP3 Inhibition for Both Synthetic Lethality and Tolerability with Chemotherapy in Preclinical Models.

The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084-94. ©2016 AACR.

Mouse and Human Genetic Analyses Associate Kalirin with Ventral Striatal Activation during Impulsivity and with Alcohol Misuse.

Impulsivity is associated with a spectrum of psychiatric disorders including drug addiction. To investigate genetic associations with impulsivity and initiation of drug taking, we took a two-step approach. First, we identified genes whose expression level in prefrontal cortex, striatum and accumbens were associated with impulsive behavior in the 5-choice serial reaction time task across 10 BXD recombinant inbred (BXD RI) mouse strains and their progenitor C57BL/6J and DBA2/J strains. Behavioral data were correlated with regional gene expression using GeneNetwork (www.genenetwork.org), to identify 44 genes whose probability of association with impulsivity exceeded a false discovery rate of < 0.05. We then interrogated the IMAGEN database of 1423 adolescents for potential associations of SNPs in human homologs of those genes identified in the mouse study, with brain activation during impulsive performance in the Monetary Incentive Delay task, and with novelty seeking scores from the Temperament and Character Inventory, as well as alcohol experience. There was a significant overall association between the human homologs of impulsivity-related genes and percentage of premature responses in the MID task and with fMRI BOLD-response in ventral striatum (VS) during reward anticipation. In contrast, no significant association was found between the polygenic scores and anterior cingulate cortex activation. Univariate association analyses revealed that the G allele (major) of the intronic SNP rs6438839 in the KALRN gene was significantly associated with increased VS activation. Additionally, the A-allele (minor) of KALRN intronic SNP rs4634050, belonging to the same haplotype block, was associated with increased frequency of binge drinking.

Versatility in phospho-dependent molecular recognition of the XRCC1 and XRCC4 DNA-damage scaffolds by aprataxin-family FHA domains.

Aprataxin, aprataxin and PNKP-like factor (APLF) and polynucleotide kinase phosphatase (PNKP) are key DNA-repair proteins with diverse functions but which all contain a homologous forkhead-associated (FHA) domain. Their primary binding targets are casein kinase 2-phosphorylated forms of the XRCC1 and XRCC4 scaffold molecules which respectively coordinate single-stranded and double-stranded DNA break repair pathways. Here, we present the high-resolution X-ray structure of a complex of phosphorylated XRCC4 with APLF, the most divergent of the three FHA domain family members. This, combined with NMR and biochemical analysis of aprataxin and APLF binding to singly and multiply-phosphorylated forms of XRCC1 and XRCC4, and comparison with PNKP reveals a pattern of distinct but overlapping binding specificities that are differentially modulated by multi-site phosphorylation. Together, our data illuminate important differences between activities of the three phospho-binding domains, in spite of a close evolutionary relationship between them.

Identification and characterisation of Schizosaccharomyces pombe cyclophilin 3, a cyclosporin A insensitive orthologue of human USA-CyP.

We have identified nine cyclophilins encoded in the genome of the fission yeast Schizosaccharomyces pombe (Sp). Cyclophilin 3 is an orthologue of hUSA-CyP, which is associated with Prp4/Prp3 in the [U4/U6.U5] snRNP complex and Prp18, both of which are components of the pre-mRNA splicing machinery. PPIase assays have shown SpCyp3 and hUSA-CyP to have comparable activity and substrate specificity, but SpCyp3 has a reduced sensitivity to CsA correlating with a difference in the catalytic site. Prp3, Prp4 and Prp18 proteins exist in S. pombe and nuclear localisation of SpCyp3 has been shown, indicating conservation of function between hUSA-CyP and SpCyp3.

One ring to bring them all--the role of Ku in mammalian non-homologous end joining.

The repair of DNA double strand breaks is essential for cell survival and several conserved pathways have evolved to ensure their rapid and efficient repair. The non-homologous end joining pathway is initiated when Ku binds to the DNA break site. Ku is an abundant nuclear heterodimer of Ku70 and Ku80 with a toroidal structure that allows the protein to slide over the broken DNA end and bind with high affinity. Once locked into placed, Ku acts as a tool-belt to recruit multiple interacting proteins, forming one or more non-homologous end joining complexes that act in a regulated manner to ensure efficient repair of DNA ends. Here we review the structure and functions of Ku and the proteins with which it interacts during non-homologous end joining.

TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function.

Topoisomerase II (TOP2) removes torsional stress from DNA and facilitates gene transcription by introducing transient DNA double-strand breaks (DSBs). Such DSBs are normally rejoined by TOP2 but on occasion can become abortive and remain unsealed. Here we identify homozygous mutations in the TDP2 gene encoding tyrosyl DNA phosphodiesterase-2, an enzyme that repairs 'abortive' TOP2-induced DSBs, in individuals with intellectual disability, seizures and ataxia. We show that cells from affected individuals are hypersensitive to TOP2-induced DSBs and that loss of TDP2 inhibits TOP2-dependent gene transcription in cultured human cells and in mouse post-mitotic neurons following abortive TOP2 activity. Notably, TDP2 is also required for normal levels of many gene transcripts in developing mouse brain, including numerous gene transcripts associated with neurological function and/or disease, and for normal interneuron density in mouse cerebellum. Collectively, these data implicate chromosome breakage by TOP2 as an endogenous threat to gene transcription and to normal neuronal development and maintenance.