PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair.

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

BRAT1 links Integrator and defective RNA processing with neurodegeneration.

Mutations in BRAT1, encoding BRCA1-associated ATM activator 1, have been associated with neurodevelopmental and neurodegenerative disorders characterized by heterogeneous phenotypes with varying levels of clinical severity. However, the underlying molecular mechanisms of disease pathology remain poorly understood. Here, we show that BRAT1 tightly interacts with INTS9/INTS11 subunits of the Integrator complex that processes 3' ends of various noncoding RNAs and pre-mRNAs. We find that Integrator functions are disrupted by BRAT1 deletion. In particular, defects in BRAT1 impede proper 3' end processing of UsnRNAs and snoRNAs, replication-dependent histone pre-mRNA processing, and alter the expression of protein-coding genes. Importantly, impairments in Integrator function are also evident in patient-derived cells from BRAT1 related neurological disease. Collectively, our data suggest that defects in BRAT1 interfere with proper Integrator functions, leading to incorrect expression of RNAs and proteins, resulting in neurodegeneration.

PARP inhibition impedes the maturation of nascent DNA strands during DNA replication.

Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1-/- cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.

XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair.

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.

XRCC1 prevents toxic PARP1 trapping during DNA base excision repair.

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 ß 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 ß 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.

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.

Neuronal enhancers are hotspots for DNA single-strand break repair.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

Pathogenic ARH3 mutations result in ADP-ribose chromatin scars during DNA strand break repair.

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.

Pathological mutations in PNKP trigger defects in DNA single-strand break repair but not DNA double-strand break repair.

Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.

ATAD5 deficiency alters DNA damage metabolism and sensitizes cells to PARP inhibition.

Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.

Temporal changes in years of life lost associated with heat waves in the Czech Republic.

Seniors constitute the population group generally most at risk of mortality due to heat stress. As life expectancy increases and health conditions of elderly people improve over time, vulnerability of the population to heat changes as well. We employed the years-of-life-lost (YLL) approach, considering life expectancy at the time of each death, to investigate how population ageing affects temporal changes in heat-related mortality in the Czech Republic. Using an updated gridded meteorological database, we identified heat waves during 1994-2017, and analysed temporal changes in their impacts on YLL and mortality. The mean impact of a heat-wave day on relative excess mortality and YLL had declined by approximately 2-3% per decade. That decline abated in the current decade, however, and the decreasing trend in mean excess mortality as well as YLL vanished when the short-term mortality displacement effect was considered. Moreover, the cumulative number of excess deaths and YLL during heat waves rose due to increasing frequency and intensity of heat waves during the examined period. The results show that in studies of temporal changes it is important to differentiate between mean effects of heat waves on mortality and the overall death burden associated with heat waves. Analysis of the average ratio of excess YLL/death per heat-wave day indicated that the major heat-vulnerable population group shifted towards older age (70+ years among males and 75+ years among females). Our findings highlight the importance of focusing heat-protection measures especially upon the elderly population, which is most heat-vulnerable and whose numbers are rising.

Homozygous pathogenic variant in BRAT1 associated with nonprogressive cerebellar ataxia.

OBJECTIVE: To investigate the pathogenicity of a novel homozygous BRAT1 variant in 2 siblings with nonprogressive cerebellar ataxia (NPCA) through functional studies on primary and immortalized patient cell lines. METHODS: BRAT1 protein levels and ataxia-telangiectasia mutated (ATM) kinase activity in patient-derived and control cell lines were assessed by Western blotting. The impact of the novel BRAT1 variants on mitochondrial function was also assessed, by comparing patient and control cell lines for rates of oxygen consumption and for phosphorylation (S293) of the E1⍺ subunit of pyruvate dehydrogenase (PDH). RESULTS: Two male siblings with NPCA, mild intellectual disability, and isolated cerebellar atrophy were found to be homozygous for a c.185T>A (p.Val62Glu) variant in BRAT1 by whole exome sequencing. Western blotting revealed markedly decreased BRAT1 protein levels in lymphocytes and/or fibroblast cells from both affected siblings compared to control cell lines. There were no differences between the patient and control cells in ATM kinase activation, following ionizing radiation. Mitochondrial studies were initially suggestive of a defect in regulation of PDH activity, but there was no evidence of increased phosphorylation of the E1⍺ subunit of the PDH complex. Measurement of oxygen consumption rates similarly failed to identify differences between patient and control cells. CONCLUSIONS: Biallelic pathogenic variants in BRAT1 can be associated with NPCA, a phenotype considerably milder than previously reported. Surprisingly, despite the molecular role currently proposed for BRAT1 in ATM regulation, this disorder is unlikely to result from defective ATM kinase or mitochondrial dysfunction.

Novel PNKP mutations causing defective DNA strand break repair and PARP1 hyperactivity in MCSZ.

OBJECTIVE: To address the relationship between novel mutations in polynucleotide 5'-kinase 3'-phosphatase (PNKP), DNA strand break repair, and neurologic disease. METHODS: We have employed whole-exome sequencing, Sanger sequencing, and molecular/cellular biology. RESULTS: We describe here a patient with microcephaly with early onset seizures (MCSZ) from the Indian sub-continent harboring 2 novel mutations in PNKP, including a pathogenic mutation in the fork-head associated domain. In addition, we confirm that MCSZ is associated with hyperactivation of the single-strand break sensor protein protein poly (ADP-ribose) polymerase 1 (PARP1) following the induction of abortive topoisomerase I activity, a source of DNA strand breakage associated previously with neurologic disease. CONCLUSIONS: These data expand the spectrum of PNKP mutations associated with MCSZ and show that PARP1 hyperactivation at unrepaired topoisomerase-induced DNA breaks is a molecular feature of this disease.

Perspectives on PARPs in S Phase.

Accurate copying of DNA during S phase is essential for genome stability and cell viability. During genome duplication, the progression of the DNA replication machinery is challenged by limitations in nucleotide supply and physical barriers in the DNA template that include naturally occurring DNA lesions and secondary structures that are difficult to replicate. To ensure correct and complete replication of the genome, cells have evolved several mechanisms that protect DNA replication forks and thus maintain genome integrity and stability during S phase. One class of enzymes that have recently emerged as important in this process, and therefore as promising targets in anticancer therapy, are the poly(ADP-ribose) polymerases (PARPs). We review here the roles of these enzymes during DNA replication as well as their impact on genome stability and cellular viability in normal and cancer cells.

PML nuclear bodies are recruited to persistent DNA damage lesions in an RNF168-53BP1 dependent manner and contribute to DNA repair.

The bulk of DNA damage caused by ionizing radiation (IR) is generally repaired within hours, yet a subset of DNA lesions may persist even for long periods of time. Such persisting IR-induced foci (pIRIF) co-associate with PML nuclear bodies (PML-NBs) and are among the characteristics of cellular senescence. Here we addressed some fundamental questions concerning the nature and determinants of this co-association, the role of PML-NBs at such sites, and the reason for the persistence of DNA damage in human primary cells. We show that the persistent DNA lesions are devoid of homologous recombination (HR) proteins BRCA1 and Rad51. Our super-resolution microscopy-based analysis showed that PML-NBs are juxtaposed to and partially overlap with the pIRIFs. Notably, depletion of 53BP1 resulted in decreased intersection between PML-NBs and pIRIFs implicating the RNF168-53BP1 pathway in their interaction. To test whether the formation and persistence of IRIFs is PML-dependent and to investigate the role of PML in the context of DNA repair and senescence, we genetically deleted PML in human hTERT-RPE-1 cells. Unexpectedly, upon high-dose IR treatment, cells displayed similar DNA damage signalling, repair dynamics and kinetics of cellular senescence regardless of the presence or absence of PML. In contrast, the PML knock-out cells showed increased sensitivity to low doses of IR and DNA-damaging agents mitomycin C, cisplatin and camptothecin that all cause DNA lesions requiring repair by HR. These results, along with enhanced sensitivity of the PML knock-out cells to DNA-PK and PARP inhibitors implicate PML as a factor contributing to HR-mediated DNA repair.

The predictability of heat-related mortality in Prague, Czech Republic, during 2015-a comparison of selected thermal indices.

We compared selected thermal indices in their ability to predict heat-related mortality in Prague, Czech Republic, during the extraordinary summer 2015. Relatively, novel thermal indices-Universal Thermal Climate Index and Excess Heat Factor (EHF)-were compared with more traditional ones (apparent temperature, simplified wet-bulb globe temperature (WBGT), and physiologically equivalent temperature). The relationships between thermal indices and all-cause relative mortality deviations from the baseline (excess mortality) were estimated by generalized additive models for the extended summer season (May-September) during 1994-2014. The resulting models were applied to predict excess mortality in 2015 based on observed meteorology, and the mortality estimates by different indices were compared. Although all predictors showed a clear association between thermal conditions and excess mortality, we found important variability in their performance. The EHF formula performed best in estimating the intensity of heat waves and magnitude of heat-impacts on excess mortality on the most extreme days. Afternoon WBGT, on the other hand, was most precise in the selection of heat-alert days during the extended summer season, mainly due to a relatively small number of "false alerts" compared to other predictors. Since the main purpose of heat warning systems is identification of days with an increased risk of heat-related death rather than prediction of exact magnitude of the excess mortality, WBGT seemed to be a slightly favorable predictor for such a system.

The Importance of Poly(ADP-Ribose) Polymerase as a Sensor of Unligated Okazaki Fragments during DNA Replication.

Poly(ADP-ribose) is synthesized by PARP enzymes during the repair of stochastic DNA breaks. Surprisingly, however, we show that most if not all endogenous poly(ADP-ribose) is detected in normal S phase cells at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or misincorporated nucleotides or from DNA replication stress. Rather, perturbation of the DNA replication proteins LIG1 or FEN1 increases S phase poly(ADP-ribose) more than 10-fold, implicating unligated Okazaki fragments as the source of S phase PARP activity. Indeed, S phase PARP activity is ablated by suppressing Okazaki fragment formation with emetine, a DNA replication inhibitor that selectively inhibits lagging strand synthesis. Importantly, PARP activation during DNA replication recruits the single-strand break repair protein XRCC1, and human cells lacking PARP activity and/or XRCC1 are hypersensitive to FEN1 perturbation. Collectively, our data indicate that PARP1 is a sensor of unligated Okazaki fragments during DNA replication and facilitates their repair.

Impacts of the 2015 Heat Waves on Mortality in the Czech Republic-A Comparison with Previous Heat Waves.

This study aimed to assess the impacts of heat waves during the summer of 2015 on mortality in the Czech Republic and to compare them with those of heat waves back to the previous record-breaking summer of 1994. We analyzed daily natural-cause mortality across the country's entire population. A mortality baseline was determined using generalized additive models adjusted for long-term trends, seasonal and weekly cycles, and identified heat waves. Mortality deviations from the baseline were calculated to quantify excess mortality during heat waves, defined as periods of at least three consecutive days with mean daily temperature higher than the 95th percentile of annual distribution. The summer of 2015 was record-breaking in the total duration of heat waves as well as their total heat load. Consequently, the impact of the major heat wave in 2015 on the increase in excess mortality relative to the baseline was greater than during the previous record-breaking heat wave in 1994 (265% vs. 240%). Excess mortality was comparable among the younger age group (0-64 years) and the elderly (65+ years) in the 1994 major heat wave while it was significantly larger among the elderly in 2015. The results suggest that the total heat load of a heat wave needs to be considered when assessing its impact on mortality, as the cumulative excess heat factor explains the magnitude of excess mortality during a heat wave better than other characteristics such as duration or average daily mean temperature during the heat wave. Comparison of the mortality impacts of the 2015 and 1994 major heat waves suggests that the recently reported decline in overall heat-related mortality in Central Europe has abated and simple extrapolation of the trend would lead to biased conclusions even for the near future. Further research is needed toward understanding the additional mitigation measures required to prevent heat-related mortality in the Czech Republic and elsewhere.

Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin.

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.

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.