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Supplementary Methods from Inhibition of ADP Ribosylation Prevents and Cures Helicobacter-Induced Gastric Preneoplasia

Supplementary Figures 1-5 from Inhibition of ADP Ribosylation Prevents and Cures Helicobacter-Induced Gastric Preneoplasia

RPA has been shown to protect single-stranded DNA (ssDNA) intermediates from instability and breakage. RPA binds ssDNA with sub-nanomolar affinity, yet dynamic turnover is required for downstream ssDNA transactions. How ultrahigh-affinity binding and dynamic turnover are achieved simultaneously is not well understood. Here we reveal that RPA has a strong propensity to assemble into dynamic condensates. In solution, purified RPA phase separates into liquid droplets with fusion and surface wetting behavior. Phase separation is stimulated by sub-stoichiometric amounts of ssDNA, but not RNA or double-stranded DNA, and ssDNA gets selectively enriched in RPA condensates. We find the RPA2 subunit required for condensation and multi-site phosphorylation of the RPA2 N-terminal intrinsically disordered region to regulate RPA self-interaction. Functionally, quantitative proximity proteomics links RPA condensation to telomere clustering and integrity in cancer cells. Collectively, our results suggest that RPA-coated ssDNA is contained in dynamic RPA condensates whose properties are important for genome organization and stability., Nature Structural & Molecular Biology, 30 (4), ISSN:1545-9993, ISSN:1545-9985

PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1.

Bloom's syndrome is caused by inactivation of the BLM helicase, which functions with TOP3A and RMI1-2 (BTR complex) to dissolve recombination intermediates and avoid somatic crossing-over. We show here that crossover avoidance by BTR further requires the activity of cyclin-dependent kinase-1 (CDK1), Polo-like kinase-1 (PLK1), and the DDR mediator protein TOPBP1, which act in the same pathway. Mechanistically, CDK1 phosphorylates BLM and TOPBP1 and promotes the interaction of both proteins with PLK1. This is amplified by the ability of TOPBP1 to facilitate phosphorylation of BLM at sites that stimulate both BLM-PLK1 and BLM-TOPBP1 binding, creating a positive feedback loop that drives rapid BLM phosphorylation at the G2-M transition. In vitro, BLM phosphorylation by CDK/PLK1/TOPBP1 stimulates the dissolution of topologically linked DNA intermediates by BLM-TOP3A. Thus, we propose that the CDK1-TOPBP1-PLK1 axis enhances BTR-mediated dissolution of recombination intermediates late in the cell cycle to suppress crossover recombination and curtail genomic instability., Science Advances, 8 (5), ISSN:2375-2548

The tumor suppressor protein BRCA1 plays an important role in DNA repair by homologous recombination. Despite being encoded by the first familial breast and ovarian cancer gene identified, how BRCA1 is recruited to sites of DNA damage to execute its repair functions has remained poorly understood. Several recent studies highlight the role of its constitutive interaction partner BARD1 in this process. In this issue, parallel work by Sherker et al (2021) focused on a second route of BRCA1 recruitment, connected to the BRCA1‐A complex protein RAP80. Studying BRCA1 recruitment in RAP80‐deficient cells exposed a critical role for the BRCA1 RING domain and its associated ubiquitin ligase activity. Given that tumors expressing RING‐less BRCA1 isoforms can become resistant to therapy, targeting the RAP80 recruitment axis in such tumors might restore effective treatment., How BRCA1 is recruited to DNA damage sites is not well understood. A study in this issue reveals that the BRCA1 RING domain and its ubiquitin ligase activity are crucial for BRCA1 recruitment in the absence of RAP80, which has potential therapeutic implications.

Centromeric integrity is key for proper chromosome segregation during cell division1. Centromeres have unique chromatin features that are essential for centromere maintenance2. Although they are intrinsically fragile and represent hotspots for chromosomal rearrangements3, little is known about how centromere integrity in response to DNA damage is preserved. DNA repair by homologous recombination requires the presence of the sister chromatid and is suppressed in the G1 phase of the cell cycle4. Here we demonstrate that DNA breaks that occur at centromeres in G1 recruit the homologous recombination machinery, despite the absence of a sister chromatid. Mechanistically, we show that the centromere-specific histone H3 variant CENP-A and its chaperone HJURP, together with dimethylation of lysine 4 in histone 3 (H3K4me2), enable a succession of events leading to the licensing of homologous recombination in G1. H3K4me2 promotes DNA-end resection by allowing DNA damage-induced centromeric transcription and increased formation of DNA–RNA hybrids. CENP-A and HJURP interact with the deubiquitinase USP11, enabling formation of the RAD51–BRCA1–BRCA2 complex5 and rendering the centromeres accessible to RAD51 recruitment and homologous recombination in G1. Finally, we show that inhibition of homologous recombination in G1 leads to centromeric instability and chromosomal translocations. Our results support a model in which licensing of homologous recombination at centromeric breaks occurs throughout the cell cycle to prevent the activation of mutagenic DNA repair pathways and preserve centromeric integrity. Centromeres are able to recruit the homologous recombination machinery during G1 via CENP-A and HJURP, thereby preserving centromeric integrity even in the absence of a sister chromatid.

Amplification of MYCN is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for MYCN-driven neuroblastoma (141 words).

Amplification of MYCN is the driving oncogenic change in a subset of high-risk neuroblastomas. The MYCN protein and the Aurora-A kinase form a complex during the S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in the S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription–replication conflicts and activates ataxia telangiectasia and Rad3-related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR kinases induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription–replication conflicts is an effective therapy for MYCN-driven neuroblastoma. Eilers and colleagues report that Aurora-A suppresses transcription–replication conflicts in MYCN-driven neuroblastoma, a vulnerability that can be targeted with a combination of Aurora-A and ATR kinase inhibitors.

Subversion of genome integrity fuels cellular adaptation and is a prerequisite for organismal evolution, yet genomic lesions are also the harmful driving force of cancer and other age-related human diseases. Genome integrity maintenance is inherently linked to genome organization and nuclear architecture, which are substantially remodeled during the cell cycle. Here we discuss recent findings on how actively dividing cells cope with endogenous genomic lesions that occur frequently at repetitive, heterochromatic, and late replicating regions as byproducts of genome duplication. We discuss how such lesions, rather than being resolved immediately when they occur, are dealt with in subsequent cell cycle phases, and even after mitotic cell division, and how this in turn affects genome organization, stability, and function.

Protein recruitment to DNA break sites is an integral part of the DNA damage response (DDR). Elucidation of the hierarchy and temporal order with which DNA damage sensors as well as repair and signaling factors assemble around chromosome breaks has painted a complex picture of tightly regulated macromolecular interactions that build specialized compartments to facilitate repair and maintenance of genome integrity. While many of the underlying interactions, e.g. between repair factors and damage-induced histone marks, can be explained by lock-and-key or induced fit binding models assuming fixed stoichiometries, structurally less well defined interactions, such as the highly dynamic multivalent interactions implicated in phase separation, also participate in the formation of multi-protein assemblies in response to genotoxic stress. Although much remains to be learned about these types of cooperative and highly dynamic interactions and their functional roles, the rapidly growing interest in material properties of biomolecular condensates and in concepts from polymer chemistry and soft matter physics to understand biological processes at different scales holds great promises. Here, we discuss nuclear condensates in the context of genome integrity maintenance, highlighting the cooperative potential between clustered stoichiometric binding and phase separation. Rather than viewing them as opposing scenarios, their combined effects can balance structural specificity with favorable physicochemical properties relevant for the regulation and function of multilayered nuclear condensates.

ADP-ribosylation, a modification of proteins, nucleic acids and metabolites, confers broad functions, including roles in stress responses elicited for example by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases, which transfer ADP-ribose from NAD(+) onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ADP-ribosyltransferases are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as anti-viral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ADP-ribosyltransferases and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ADP-ribosyltransferases that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ADP-ribosyltransferases to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.

The paradigm that checkpoints halt cell cycle progression for genome repair has been challenged by the recent discovery of heritable DNA lesions escaping checkpoint control. How such inherited lesions affect genome function and integrity is not well understood. Here, we identify a new class of heritable DNA lesions, which is marked by replication protein A (RPA), a protein primarily known for shielding single-stranded DNA in S/G2. We demonstrate that post-mitotic RPA foci occur at low frequency during unperturbed cell cycle progression, originate from the previous cell cycle, and are exacerbated upon replication stress. RPA-marked inherited ssDNA lesions are found at telomeres, particularly of ALT-positive cancer cells. We reveal that RPA protects these replication remnants in G1 to allow for post-mitotic DNA synthesis (post-MiDAS). Given that ALT-positive cancer cells exhibit high levels of replication stress and telomere fragility, targeting post-MiDAS might be a new therapeutic opportunity., Single-stranded DNA during DNA replication and repair in S/G2 needs protection by replication protein A (RPA). Here the authors reveal that RPA also shields inherited single-stranded DNA in G1, representing replication remnants from the previous cell cycle, to allow for post-mitotic DNA synthesis.

DNA double-strand breaks can be repaired by two competing mechanisms, non-homologous end-joining (NHEJ) and homologous recombination (HR). Whether one or the other repair pathway is favored depends on the availability of an undamaged template DNA that allows for homology-directed repair. The tumor suppressor proteins 53BP1 and BRCA1 are considered antagonistic players in this repair pathway choice, as 53BP1 restrains DNA end resection, whereas BRCA1, together with its partner protein BARD1, displaces 53BP1 from damaged replicated chromatin and promotes HR. How cells switch from a 53BP1-dominated to a BRCA1-dominated response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells, in comparison to 53BP1 accumulation at damaged unreplicated chromatin. These findings substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, e.g. to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

Chromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled ‘chromatin breathing’ upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails., Chromatin is dynamically remodeled in response to DNA damage in favour of repair. Here the authors reveal how the chromatin remodeler CHD7 and chromatin binding protein 53BP1 regulate distinct DNA repair pathways.

Automated high-throughput microscopy has been instrumental for drug discovery and large-scale gene perturbation screens. Advancements in image resolution, microscope speed, and detection sensitivity have greatly aided high-content imaging approaches. Here, we describe how high-content microscopy can be repurposed for quantitative image-based cytometry, an approach that exploits both the throughput and the resolution of current screening microscopes for multidimensional cell population analyses.

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.

The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9‐mediated endogenous protein tagging, that 53BP1‐marked repair compartments are dynamic, show droplet‐like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light‐induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1‐dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53‐dependent gene activation and cell fate decisions.

The G2/M checkpoint coordinates DNA replication with mitosis and thereby prevents chromosome segregation in the presence of unreplicated or damaged DNA. Here, we show that the RNA‐binding protein TIAR is essential for the G2/M checkpoint and that TIAR accumulates in nuclear foci in late G2 and prophase in cells suffering from replication stress. These foci, which we named G2/M transition granules (GMGs), occur at low levels in normally cycling cells and are strongly induced by replication stress. In addition to replication stress response proteins, GMGs contain factors involved in RNA metabolism as well as CDK1. Depletion of TIAR accelerates mitotic entry and leads to chromosomal instability in response to replication stress, in a manner that can be alleviated by the concomitant depletion of Cdc25B or inhibition of CDK1. Since TIAR retains CDK1 in GMGs and attenuates CDK1 activity, we propose that the assembly of GMGs may represent a so far unrecognized mechanism that contributes to the activation of the G2/M checkpoint in mammalian cells.

This study demonstrates how single cell normalization to genome size provides insight into genome function, here in the context of DNA double-strand break repair by 53BP1 versus BRCA1–BARD1., DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells. Our results substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1–BARD1–dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1–BARD1–mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

Summary In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk., Graphical Abstract, Highlights • Mitochondrial ADP-ribosylation was identified by different methods • Mitochondrial ADP-ribosylation reversibly increased after respiratory chain inhibition • H2O2 treatment induces nuclear and reduces mitochondrial ADP-ribosylation • Elevated mitochondrial ADP-ribosylation dampened MMS-induced ARTD1 chromatin retention, Hopp et al. detect mitochondrial ADP-ribosylation and characterize its dependency on intracellular NAD+ homeostasis. While respiratory chain inhibition increases mitochondrial ADP-ribosylation, hydrogen peroxide treatment reduces mitochondrial ADP-ribosylation and reciprocally induces nuclear ADP-ribosylation. This dynamic and reversable process is dependent on a NAD+-dependent mitochondrial-nuclear crosstalk.

A posttranslational protein modification with increasing relevance for cancer biology is poly(ADP-ribosyl)ation (PARylation) (1). Inhibitors of PARylation show promising effects either as monotherapeutic agents or as chemo- or radiosensitizers to support classical DNA damaging cancer therapies. The targets of these inhibitors are enzymes of the family of poly(ADP-ribose) polymerases (PARPs, also known as ARTDs) (2). PARP inhibitors are analogs of NAD+, which PARP enzymes use as substrate to synthesize poly(ADP-ribose) (PAR). PAR can be covalently attached to or interact non-covalently with target proteins (3,4). In humans, 17 genes encode for PARP/ARTD enzymes, of which 5 can generate PAR (PARP1/2/4 and TNKS1/2), while all others either catalyze mono-ADP-ribosylation (MARylation) or appear to be inactive. PARPs localize to various cellular compartments and participate in a multitude of cancer-relevant cellular functions, such as DNA damage responses, transcription, chromatin organization and regulation of cell death (1).

Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52. Thus, rather than suppressing HDR, 53BP1 fosters its fidelity. These findings illuminate causes and consequences of synthetic viability acquired through 53BP1 silencing in cells lacking the BRCA1 tumor suppressor. We show that such cells survive DSB assaults at the cost of increasing reliance on RAD52-mediated HDR, which may fuel genome instability. However, our findings suggest that when challenged by DSBs, BRCA1- and 53BP1-deficient cells may become hypersensitive to, and be eliminated by, RAD52 inhibition.

The p53-Mdm2 system is key to tumor suppression. We have recently reported that p53 as well as Mdm2 are capable of supporting DNA replication fork progression. On the other hand, we found that Mdm2 is a modifier of chromatin, modulating polycomb repressor complex (PRC)-driven histone modifications. Here we show that, similar to Mdm2 knockdown, the depletion of PRC members impairs DNA synthesis, as determined in fiber assays. In particular, the ubiquitin ligase and PRC1 component RNF2/Ring1B is required to support DNA replication, similar to Mdm2. Moreover, the Ring finger domain of Mdm2 is not only essential for its ubiquitin ligase activity, but also for proper DNA replication. Strikingly, Mdm2 overexpression can rescue RNF2 depletion with regard to DNA replication fork progression, and vice versa, strongly suggesting that the two ubiquitin ligases perform overlapping functions in this context. H2A overexpression also rescues fork progression upon depletion of Mdm2 or RNF2, but only when the ubiquitination sites K118/K119 are present. Depleting the H2A deubiquitinating enzyme BAP1 reduces the fork rate, suggesting that both ubiquitination and deubiquitination of H2A are required to support fork progression. The depletion of Mdm2 elicits the accumulation of RNA/DNA hybrids, suggesting R-loop formation as a mechanism of impaired DNA replication. Accordingly, RNase H overexpression or the inhibition of the transcription elongation kinase CDK9 each rescues DNA replication upon depletion of Mdm2 or RNF2. Taken together, our results suggest that chromatin modification by Mdm2 and PRC1 ensures smooth DNA replication through the avoidance of R-loop formation.

Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID. Here we showed that the HUWE1 expression is altered and mutation frequency increased in three different XLID individual (HUWE1 p.R2981H, p.R4187C and HUWE1 duplication) cell lines. The effect was most prominent in HUWE1 p.R4187C XLID cells and was accompanied with decreased DNA repair capacity and hypersensitivity to oxidative stress. Analysis of HUWE1 substrates revealed XLID-specific down-regulation of oxidative stress response DNA polymerase (Pol) λ caused by hyperactive HUWE1 p.R4187C. The subsequent restoration of Polλ levels counteracted the oxidative hypersensitivity. The observed alterations in the genome integrity maintenance may be particularly relevant in the cortical progenitor zones of human brain, as suggested by HUWE1 immunofluorescence analysis of cerebral organoids. These results provide evidence that impairments of the fundamental cellular processes, like genome integrity maintenance, characterize HUWE1-promoted XLID.

Replication stress is a major source of DNA damage and an important driver of cancer development. Replication intermediates that occur upon mild forms of replication stress frequently escape cell cycle checkpoints and can be transmitted through mitosis into the next cell cycle. The consequences of such inherited DNA lesions for cell fate and survival are poorly understood. By using time-lapse microscopy and quantitative image-based cytometry to simultaneously monitor inherited DNA lesions marked by the genome caretaker protein 53BP1 and cell cycle progression, we show that inheritance of 53BP1-marked lesions from the previous S-phase is associated with a prolonged G1 duration in the next cell cycle. These results suggest that cell-to-cell variation in S-phase commitment is determined, at least partially, by the amount of replication-born inherited DNA damage in individual cells. We further show that loss of the tumor suppressor protein p53 overrides replication stress-induced G1 prolongation and allows S-phase entry with excessive amounts of inherited DNA lesions. Thus, replication stress and p53 loss may synergize during cancer development by promoting cell cycle re-entry with unrepaired mutagenic DNA lesions originating from the previous cell cycle.

Protecting stalled DNA replication forks from degradation by promiscuous nucleases is essential to prevent genomic instability, a major driving force of tumorigenesis. Several proteins commonly associated with the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) have been implicated in the stabilization of stalled forks. Human CtIP, in conjunction with the MRE11 nuclease complex, plays an important role in HR by promoting DSB resection. Here, we report an unanticipated function for CtIP in protecting reversed forks from degradation. Unlike BRCA proteins, which defend nascent DNA strands from nucleolytic attack by MRE11, we find that CtIP protects perturbed forks from erroneous over-resection by DNA2. Finally, we uncover functionally synergistic effects between CtIP and BRCA1 in mitigating replication-stress-induced genomic instability. Collectively, our findings reveal a DSB-resection- and MRE11-independent role for CtIP in preserving fork integrity that contributes to the survival of BRCA1-deficient cells.

Herein, we introduce microfluidic superheating as a new method for peptide fragmentation prior to mass spectrometric analysis. The superheating conditions were found to be stable up to 240 °C for more than 30 min without elevated pressure or boiling of the aqueous sample. As proof of principle, we exposed the peptides ACTH1-10 and OVA257-264 to various superheating conditions, causing different degrees of decomposition. Optimized superheating conditions resulted in the entire peptide ladder sequence of the y-ions, allowing the amino acid sequence to be deduced from a single-stage mass spectrum. Thus, obtaining information in the same quality as from tandem mass spectrometry can be achieved by a single superheating step.

Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID. Here we showed that the HUWE1 expression is altered and mutation frequency increased in three different XLID individual (HUWE1 p.R2981H, p.R4187C and HUWE1 duplication) cell lines. The effect was most prominent in HUWE1 p.R4187C XLID cells and was accompanied with decreased DNA repair capacity and hypersensitivity to oxidative stress. Analysis of HUWE1 substrates revealed XLID-specific down-regulation of oxidative stress response DNA polymerase (Pol) λ caused by hyperactive HUWE1 p.R4187C. The subsequent restoration of Polλ levels counteracted the oxidative hypersensitivity. The observed alterations in the genome integrity maintenance may be particularly relevant in the cortical progenitor zones of human brain, as suggested by HUWE1 immunofluorescence analysis of cerebral organoids. These results provide evidence that impairments of the fundamental cellular processes, like genome integrity maintenance, characterize HUWE1-promoted XLID. © The Author(s) 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Exploiting the full potential of anti-cancer drugs necessitates a detailed understanding of their cytotoxic effects. While standard omics approaches are limited to cell population averages, emerging single cell techniques currently lack throughput and are not applicable for compound screens. Here, we employed a versatile and sensitive high-content microscopy-based approach to overcome these limitations and quantify multiple parameters of cytotoxicity at the single cell level and in a cell cycle resolved manner. Applied to PARP inhibitors (PARPi) this approach revealed an S-phase-specific DNA damage response after only 15 min, quantitatively differentiated responses to several clinically important PARPi, allowed for cell cycle resolved analyses of PARP trapping, and predicted conditions of PARPi hypersensitivity and resistance. The approach illuminates cellular mechanisms of drug synergism and, through a targeted multivariate screen, could identify a functional interaction between PARPi olaparib and NEDD8/SCF inhibition, which we show is dependent on PARP1 and linked to PARP1 trapping., Methods to study anti-cancer drugs cytotoxicity are often low throughput and rely on population average. Here the authors present an automated image-based cytometry method to quantify multiple cytotoxicity parameters in single cells, and use it to study the effect of PARP inhibitors in cancer cells.

Formation of poly(ADP-ribose) (PAR) marks intracellular stress signaling and is notably induced upon DNA damage. PAR polymerases (PARPs) catalyze PAR synthesis upon genotoxic stress and thereby recruit multiple proteins to damaged chromatin. PAR induction is transient and antagonized by the action of PAR glycohydrolase (PARG). Given that poly(ADP-ribosyl)ation (PARylation) is involved in genome integrity maintenance and other vital cellular functions, but also in light of the recent approval of PARP inhibitors for cancer treatments, reliable measurements of intracellular PAR formation have gained importance. Here we provide a detailed protocol for PAR measurements by quantitative image-based cytometry. This technique combines the high spatial resolution of single-cell microscopy with the advantages of cell population measurements through automated high-content imaging. Such upscaling of immunofluorescence-based PAR detection not only increases the robustness of the measurements through averaging across large cell populations but also allows for the discrimination of subpopulations and thus enables multivariate measurements of PAR levels and DNA damage signaling. We illustrate how this technique can be used to assess the dynamics of the cellular response to oxidative damage as well as to PARP inhibitor-induced genotoxicity in a cell cycle resolved manner. Due to the possibility to use any automated microscope for quantitative image-based cytometry, the presented method has widespread applicability in the area of PARP biology and beyond.

Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling.