Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells.

Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation.

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.

TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs.

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.

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation.

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.

Guarding against collateral damage during chromatin transactions.

Signal amplifications are vital for chromatin function, yet they also bear the risk of transforming into unrestrained, self-escalating, and potentially harmful responses. Examples of inbuilt limitations are emerging, revealing how chromatin transactions are confined within physiological boundaries.

ATR prohibits replication catastrophe by preventing global exhaustion of RPA.

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors.

Ubiquitin Phosphorylation at Thr12 Modulates the DNA Damage Response.

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.

TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.

Activation of homologous recombination in G1 preserves centromeric integrity.

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.

Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability.

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.

CtIP-Mediated Fork Protection Synergizes with BRCA1 to Suppress Genomic Instability upon DNA Replication Stress.

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.

The Hammer and the Dance of Cell Cycle Control.

Cell cycle checkpoints secure ordered progression from one cell cycle phase to the next. They are important to signal cell stress and DNA lesions and to stop cell cycle progression when severe problems occur. Recent work suggests, however, that the cell cycle control machinery responds in more subtle and sophisticated ways when cells are faced with naturally occurring challenges, such as replication impediments associated with endogenous replication stress. Instead of following a stop and go approach, cells use fine-tuned deceleration and brake release mechanisms under the control of ataxia telangiectasia and Rad3-related protein kinase (ATR) and checkpoint kinase 1 (CHK1) to more flexibly adapt their cell cycle program to changing conditions. We highlight emerging examples of such intrinsic cell cycle checkpoint regulation and discuss their physiological and clinical relevance.

Genome maintenance meets mechanobiology.

Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark of aging and of age-associated diseases including cancer and neurodegeneration. To maintain a stable genome, genome surveillance and repair pathways are closely intertwined with cell cycle regulation and with DNA transactions that occur during transcription and DNA replication. Coordination of these processes across different time and length scales involves dynamic changes of chromatin topology, clustering of fragile genomic regions and repair factors into nuclear repair centers, mobilization of the nuclear cytoskeleton, and activation of cell cycle checkpoints. Here, we provide a general overview of cell cycle regulation and of the processes involved in genome duplication in human cells, followed by an introduction to replication stress and to the cellular responses elicited by perturbed DNA synthesis. We discuss fragile genomic regions that experience high levels of replication stress, with a particular focus on telomere fragility caused by replication stress at the ends of linear chromosomes. Using alternative lengthening of telomeres (ALT) in cancer cells and ALT-associated PML bodies (APBs) as examples of replication stress-associated clustered DNA damage, we discuss compartmentalization of DNA repair reactions and the role of protein properties implicated in phase separation. Finally, we highlight emerging connections between DNA repair and mechanobiology and discuss how biomolecular condensates, components of the nuclear cytoskeleton, and interfaces between membrane-bound organelles and membraneless macromolecular condensates may cooperate to coordinate genome maintenance in space and time.

Poly(ADP-ribose) polymerase 1 participates in the phase entrainment of circadian clocks to feeding.

Circadian clocks in peripheral organs are tightly coupled to cellular metabolism and are readily entrained by feeding-fasting cycles. However, the molecular mechanisms involved are largely unknown. Here we show that in liver the activity of PARP-1, an NAD(+)-dependent ADP-ribosyltransferase, oscillates in a daily manner and is regulated by feeding. We provide biochemical evidence that PARP-1 binds and poly(ADP-ribosyl)ates CLOCK at the beginning of the light phase. The loss of PARP-1 enhances the binding of CLOCK-BMAL1 to DNA and leads to a phase-shift of the interaction of CLOCK-BMAL1 with PER and CRY repressor proteins. As a consequence, CLOCK-BMAL1-dependent gene expression is altered in PARP-1-deficient mice, in particular in response to changes in feeding times. Our results show that Parp-1 knockout mice exhibit impaired food entrainment of peripheral circadian clocks and support a role for PARP-1 in connecting feeding with the mammalian timing system.

PARP1 proximity proteomics reveals interaction partners at stressed replication forks.

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.

Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments.

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.

Analysis of the PARP1, ADP-Ribosylation, and TRIP12 Triad With Markers of Patient Outcome in Human Breast Cancer.

PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses and PARPi resistance with relapse currently pose limitations to the efficacy of PARPi therapy. The pathobiological reasons why individual patients respond differently to PARPi are poorly understood. In this study, we analyzed expression of PARP1, the main target of PARPi, in normal breast tissue, breast cancer, and its precursor lesions using human breast cancer tissue microarrays covering a total of 824 patients, including more than 100 TNBC cases. In parallel, we analyzed nuclear adenosine diphosphate (ADP)-ribosylation as a marker of PARP1 activity and TRIP12, an antagonist of PARPi-induced PARP1 trapping. Although we found PARP1 expression to be generally increased in invasive breast cancer, PARP1 protein levels and nuclear ADP-ribosylation were lower in higher tumor grade and TNBC samples than non-TNBCs. Cancers with low levels of PARP1 and low levels of nuclear ADP-ribosylation were associated with significantly reduced overall survival. This effect was even more pronounced in cases with high levels of TRIP12. These results indicate that PARP1-dependent DNA repair capacity may be compromised in aggressive breast cancers, potentially fueling enhanced accumulation of mutations. Moreover, the results revealed a subset of breast cancers with low PARP1, low nuclear ADP-ribosylation, and high TRIP12 levels, which may compromise their response to PARPi, suggesting a combination of markers for PARP1 abundance, enzymatic activity, and trapping capabilities might aid patient stratification for PARPi therapy.

When the RAP (80) fades out, you can hear BRCA1 RING.

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.

Proteome-wide identification of poly(ADP-Ribosyl)ation targets in different genotoxic stress responses.

Poly(ADP-ribos)ylation (PARylation) is a reversible posttranslational modification found in higher eukaryotes. However, little is known about PARylation acceptor proteins. Here, we describe a sensitive proteomics approach based on high-accuracy quantitative mass spectrometry for the identification of PARylated proteins induced under different cellular stress conditions. While confirming the majority of known PARylated substrates, our screen identifies numerous additional PARylation targets. In vivo and in vitro validation of acceptor proteins confirms that our methodology targets covalent PARylation. Nuclear proteins encompassing nucleic acid binding properties are prominently PARylated upon genotoxic stress, consistent with the nuclear localization of ARTD1/PARP1 and ARTD2/PARP2. Distinct differences in proteins becoming PARylated upon various genotoxic insults are observed, exemplified by the PARylation of RNA-processing factors THRAP3 and TAF15 under oxidative stress. High-content imaging reveals that PARylation affects the nuclear relocalization of THRAP3 and TAF15, demonstrating the potential of our approach to uncover hitherto unappreciated processes being controlled by specific genotoxic-stress-induced PARylation.

The chromatin scaffold protein SAFB1 renders chromatin permissive for DNA damage signaling.

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.