Repair Pathway Choice Research Articles

DTX3L-mediated TIRR nuclear export and degradation regulates DNA repair pathway choice and PARP inhibitor sensitivity

53BP1 plays an important role in DNA double-strand break (DSB) repair and this activity is negatively regulated by its interaction with Tudor interacting repair regulator (TIRR). However, how the TIRR-53BP1 repair axis is regulated in response to DNA damage remains elusive. Here, we demonstrate that TIRR is translocated to the cytoplasm and degraded upon DNA damage. Ubiquitination of TIRR at lysine 187 by DTX3L is a critical process that regulates NHEJ pathway activity and PARP inhibitor sensitivity by facilitating XPO1-mediated TIRR nuclear export and degradation after DNA damage. We show that DTX3L is overexpressed in prostate cancers in patients and that decreased expression of TIRR due to DTX3L overexpression impairs the negative regulatory effect of TIRR on 53BP1, which consequently induces HR deficiency and chromosomal instability and sensitizes prostate cancer cells to poly (ADP-ribose) polymerase (PARP) inhibitors. Our work reveals a dual action of DTX3L on TIRR degradation and nuclear exportation and identifies DTX3L as an upstream regulator of the TIRR-53BP1 axis that governs DNA repair pathway choice and PARP inhibitor sensitivity. These findings suggest that TIRR ubiquitination and DTX3L overexpression could be viable biomarkers predicting PARP inhibitor sensitivity in cancers.

Saccharomyces cerevisiae Rev7 promotes non-homologous end-joining by blocking Mre11 nuclease and Rad50's ATPase activities and homologous recombination.

Recent studies have shown that, in human cancer cells, the tetrameric Shieldin complex (comprising REV7, SHLD1, SHLD2, and SHLD3) facilitates non-homologous end-joining (NHEJ) while blocking homologous recombination (HR). Surprisingly, several eukaryotic species lack SHLD1, SHLD2, and SHLD3 orthologs, suggesting that Rev7 may leverage an alternative mechanism to regulate the double-strand break (DSB) repair pathway choice. Exploring this hypothesis, we discovered that Saccharomyces cerevisiae Rev7 physically interacts with the Mre11-Rad50-Xrs2 (MRX) subunits, impedes G-quadruplex DNA synergized HU-induced toxicity, and facilitates NHEJ, while antagonizing HR. Notably, we reveal that a 42-amino acid C-terminal fragment of Rev7 binds to the subunits of MRX complex, protects rev7∆ cells from G-quadruplex DNA-HU-induced toxicity, and promotes NHEJ by blocking HR. By comparison, the N-terminal HORMA domain, a conserved protein-protein interaction module, was dispensable. We further show that the full-length Rev7 impedes Mre11 nuclease and Rad50's ATPase activities without affecting the latter's ATP-binding ability. Combined, these results provide unanticipated insights into the functional interaction between the MRX subunits and Rev7 and highlight a previously unrecognized mechanism by which Rev7 facilitates DSB repair via NHEJ, and attenuation of HR, by blocking Mre11 nuclease and Rad50's ATPase activities in S. cerevisiae.

53BP1-the ‘Pandora’s box’ of genome integrity

53BP1 has several functions in the maintenance of genome integrity. It functions as a key mediator involved in double-strand break (DSB) repair, which functions to maintain a balance in the repair pathway choices and in preserving genomic stability. While its DSB repair functions are relatively well-characterized, its role in DNA replication and replication fork protection is less understood. In response to replication stress, 53BP1 contributes to fork protection by regulating fork reversal and restart. It helps maintain replication fork stability and speed, with 53BP1 loss leading to defective fork progression and increased sensitivity to replication stress agents. However, 53BP1's precise role in fork protection remains debated, as some studies have not observed protective effects. Therefore, it is critical to determine the role of 53BP1 in replication to better understand when it promotes replication fork protection, and the underlying mechanisms involved. Moreover, 53BP1's function in replication stress extends beyond its activity at active replication forks; it also forms specialized nuclear bodies (NBs) which protect stretches of under-replicated DNA (UR-DNA) transmitted from a previous cell cycle to daughter cells through mitosis. The mechanism of 53BP1 NBs in the coordination of replication and repair events at UR-DNA loci is not fully understood and warrants further investigation. The present review article focuses on elucidating 53BP1’s functions in replication stress (RS), its role in replication fork protection, and the significance of 53BP1 NBs in this context to provide a more comprehensive understanding of its less well-established role in DNA replication.

TPX2 serves as a novel target for expanding the utility of PARPi in pancreatic cancer through conferring synthetic lethality

BackgroundPARP inhibitors (PARPi) have been licensed for the maintenance therapy of patients with metastatic pancreatic cancer carrying pathogenic germline BRCA1/2 mutations. However, mutations in BRCA1/2 are notably rare in pancreatic...

One-ended and two-ended breaks at nickase-broken replication forks

Replisome collision with a nicked parental DNA template can lead to the formation of a replication-associated double strand break (DSB). How this break is repaired has implications for cancer initiation, cancer therapy and therapeutic gene editing. Recent work shows that collision of a replisome with a nicked DNA template can give rise to either a single-ended (se) or a double-ended (de)DSB, with potentially divergent effects on repair pathway choice and genomic instability. Emerging evidence suggests that the biochemical environment of the broken mammalian replication fork may be specialized in such a way as to skew repair in favor of homologous recombination at the expense of non-homologous end joining.

The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

Shieldin and CST co-orchestrate DNA polymerase-dependent tailed-end joining reactions independently of 53BP1-governed repair pathway choice.

Tumor suppressor p53-binding protein 1 (53BP1) regulates DNA end joining in lymphocytes, diversifying immune antigen receptors. This involves nucleosome-bound 53BP1 at DNA double-stranded breaks (DSBs) recruiting Rap1-interacting factor 1 homolog (RIF1) and shieldin, a poorly understood DNA-binding complex. The 53BP1-RIF1-shieldin axis is pathological in BRCA1-mutated cancers, blocking homologous recombination (HR) and driving illegitimate nonhomologous end joining (NHEJ). However, how this axis regulates DNA end joining and HR suppression remains unresolved. We investigated shieldin and its interplay with the Ctc1-Stn1-Ten1 (CST) complex, which was recently implicated downstream of 53BP1. Immunophenotypically, mice lacking shieldin or CST are equivalent, with class-switch recombination coreliant on both complexes. Ataxia-telangiectasia mutated kinase-dependent DNA damage signaling underpins this cooperation, inducing physical interactions between these complexes that reveal shieldin as a DSB-responsive CST adaptor. Furthermore, DNA polymerase ζ functions downstream of shieldin, establishing DNA fill-in synthesis as the physiological function of shieldin-CST. Lastly, we demonstrate that 53BP1 suppresses HR and promotes NHEJ in BRCA1-deficient mice and cells independently of shieldin. These findings showcase the versatility of the 53BP1 pathway, achieved through the collaboration of chromatin-bound 53BP1 complexes and DNA end-processing effector proteins.

Deletion of IRC19 Causes Defects in DNA Double-Strand Break Repair Pathways in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair is a fundamental cellular process crucial for maintaining genome stability, with homologous recombination and non-homologous end joining as the primary mechanisms, and various alternative pathways such as single-strand annealing (SSA) and microhomology-mediated end joining also playing significant roles under specific conditions. IRC genes were previously identified as part of a group of genes associated with increased levels of Rad52 foci in Saccharomyces cerevisiae. In this study, we investigated the effects of IRC gene mutations on DSB repair, focusing on uncharacterized IRC10, 19, 21, 22, 23, and 24. Gene conversion (GC) assay revealed that irc10Δ, 22Δ, 23Δ, and 24Δ mutants displayed modest increases in GC frequencies, while irc19Δ and irc21Δ mutants exhibited significant reductions. Further investigation revealed that deletion mutations in URA3 were not generated in irc19Δ mutant cells following HO-induced DSBs. Additionally, irc19Δ significantly reduced frequency of SSA, and a synergistic interaction between irc19Δ and rad52Δ was observed in DSB repair via SSA. Assays to determine the choice of DSB repair pathways indicated that Irc19 is necessary for generating both GC and deletion products. Overall, these results suggest a potential role of Irc19 in DSB repair pathways, particularly in end resection process.

The type of DNA damage response after decitabine treatment depends on the level of DNMT activity.

Decitabine and azacytidine are considered as epigenetic drugs that induce DNA methyltransferase (DNMT)-DNA crosslinks, resulting in DNA hypomethylation and damage. Although they are already applied against myeloid cancers, important aspects of their mode of action remain unknown, highly limiting their clinical potential. Using a combinatorial approach, we reveal that the efficacy profile of both compounds primarily depends on the level of induced DNA damage. Under low DNMT activity, only decitabine has a substantial impact. Conversely, when DNMT activity is high, toxicity and cellular response to both compounds are dramatically increased, but do not primarily depend on DNA hypomethylation or RNA-associated processes. By investigating proteome dynamics on chromatin, we show that decitabine induces a strictly DNMT-dependent multifaceted DNA damage response based on chromatin recruitment, but not expression-level changes of repair-associated proteins. The choice of DNA repair pathway hereby depends on the severity of decitabine-induced DNA lesions. Although under moderate DNMT activity, mismatch (MMR), base excision (BER), and Fanconi anaemia-dependent DNA repair combined with homologous recombination are activated in response to decitabine, high DNMT activity and therefore immense replication stress induce activation of MMR and BER followed by non-homologous end joining.

Histone H2A variants play a key role at DNA double-strand breaks during repair pathway choice

Histone H2A variants play a key role at DNA double-strand breaks during repair pathway choice

Repetitive Sequence Stability in Embryonic Stem Cells.

Repetitive sequences play an indispensable role in gene expression, transcriptional regulation, and chromosome arrangements through trans and cis regulation. In this review, focusing on recent advances, we summarize the epigenetic regulatory mechanisms of repetitive sequences in embryonic stem cells. We aim to bridge the knowledge gap by discussing DNA damage repair pathway choices on repetitive sequences and summarizing the significance of chromatin organization on repetitive sequences in response to DNA damage. By consolidating these insights, we underscore the critical relationship between the stability of repetitive sequences and early embryonic development, seeking to provide a deeper understanding of repetitive sequence stability and setting the stage for further research and potential therapeutic strategies in developmental biology and regenerative medicine.

Profiling Cas9- and Cas12a-induced mutagenesis in Arabidopsis thaliana.

With the advancement of CRISPR technologies, a comprehensive understanding of repair mechanisms following double-strand break (DSB) formation is important for improving the precision and efficiency of genetic modifications. In plant genetics, two Cas nucleases are widely used, i.e. Cas9 and Cas12a, which differ with respect to PAM sequence composition, position of the DSB relative to the PAM, and DSB-end configuration (blunt vs. staggered). The latter difference has led to speculations about different options for repair and recombination. Here, we provide detailed repair profiles for LbCas12a in Arabidopsis thaliana, using identical experimental settings previously reported for Cas9-induced DSBs, thus allowing for a quantitative comparison of both nucleases. For both enzymes, non-homologous end-joining (NHEJ) produces 70% of mutations, whereas polymerase theta-mediated end-joining (TMEJ) generates 30%, indicating that DSB-end configuration does not dictate repair pathway choice. Relevant for genome engineering approaches aimed at integrating exogenous DNA, we found that Cas12a similarly stimulates the integration of T-DNA molecules as does Cas9. Long-read sequencing of both Cas9 and Cas12a repair outcomes further revealed a previously underappreciated degree of DNA loss upon TMEJ. The most notable disparity between Cas9 and Cas12a repair profiles is caused by how NHEJ acts on DSB ends with short overhangs: non-symmetric Cas9 cleavage produce 1 bp insertions, which we here show to depend on polymerase Lambda, whereas staggered Cas12a DSBs are not subjected to fill-in synthesis. We conclude that Cas9 and Cas12a are equally effective for genome engineering purposes, offering flexibility in nuclease choice based on the availability of compatible PAM sequences.

Current trends and future perspectives in hadron therapy: radiobiology

PurposeThe purpose of this article was to highlight current and future trends in radiobiology in an effort to move hadron therapy forward through the application of new knowledge in DNA damage and subsequent response to heavy ion radiotherapy, immune oncology and the interconnection between.MethodsThe subject matter begins with a description of the role of radiation in eliciting either an immunogenic or tolerogenic response to radiation exposure. The role of fragmented DNA in an immunogenic response is described, followed by the definitive role that DNA damage and subsequent repair, or not, of complex DNA damage after hadron exposure plays in the survival response of hadron irradiated cells.ResultsThe process by which ionizing radiation elicits an immunogenic rather than tolerogenic response is becoming clearer. The timing of fractionated radiotherapy when combined with an immune checkpoint inhibitor is not clear and may be tumor site specific. Furthermore, whether hadron therapy is more effective at generating a durable immunogenic response is unknown.ConclusionsCytosolic DNA plays a significant role in eliciting an innate immune response with the likelihood that hadron therapy would generate complex DNA damage that because it is less likely to be repaired, is more likely to become cytosolic DNA, and more likely to activate an immunogenic response. Lastly, DNA repair pathway choice appears to be a credible bio-indicator for hadron therapy selection as well identify druggable targets to enhance hadron therapy.

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

An E3 ubiquitin ligase localization screen uncovers DTX2 as a novel ADP-ribosylation-dependent regulator of DNA double-strand break repair

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: non-homologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response (DDR) relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DDR regulation remains incomplete.Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Amongst these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DTC domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 re-expression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

Potential Benefits of Combining Proton or Carbon Ion Therapy with DNA Damage Repair Inhibitors.

The use of charged particle radiotherapy is currently increasing, but combination therapy with DNA repair inhibitors remains to be exploited in the clinic. The high-linear energy transfer (LET) radiation delivered by charged particles causes clustered DNA damage, which is particularly effective in destroying cancer cells. Whether the DNA damage response to this type of damage is different from that elicited in response to low-LET radiation, and if and how it can be targeted to increase treatment efficacy, is not fully understood. Although several preclinical studies have reported radiosensitizing effects when proton or carbon ion irradiation is combined with inhibitors of, e.g., PARP, ATR, ATM, or DNA-PKcs, further exploration is required to determine the most effective treatments. Here, we examine what is known about repair pathway choice in response to high- versus low-LET irradiation, and we discuss the effects of inhibitors of these pathways when combined with protons and carbon ions. Additionally, we explore the potential effects of DNA repair inhibitors on antitumor immune signaling upon proton and carbon ion irradiation. Due to the reduced effect on healthy tissue and better immune preservation, particle therapy may be particularly well suited for combination with DNA repair inhibitors.

The chromatin-associated 53BP1 ortholog, HSR-9, regulates recombinational repair and X chromosome segregation in the Caenorhabditis elegans germ line.

53BP1 plays a crucial role in regulating DNA damage repair pathway choice and checkpoint signaling in somatic cells; however, its role in meiosis has remained enigmatic. In this study, we demonstrate that the Caenorhabditis elegans ortholog of 53BP1, HSR-9, associates with chromatin in both proliferating and meiotic germ cells. Notably, HSR-9 is enriched on the X chromosome pair in pachytene oogenic germ cells. HSR-9 is also present at kinetochores during both mitotic and meiotic divisions but does not appear to be essential for monitoring microtubule-kinetochore attachments or tension. Using cytological markers of different steps in recombinational repair, we found that HSR-9 influences the processing of a subset of meiotic double-stranded breaks into COSA-1-marked crossovers. Additionally, HSR-9 plays a role in meiotic X chromosome segregation under conditions where X chromosomes fail to pair, synapse, and recombine. Together, these results highlight that chromatin-associated HSR-9 has both conserved and unique functions in the regulation of meiotic chromosome behavior.

New facets in the chromatin-based regulation of genome maintenance

The maintenance of genome integrity by DNA damage response machineries is key to protect cells against pathological development. In cell nuclei, these genome maintenance machineries operate in the context of chromatin, where the DNA wraps around histone proteins. Here, we review recent findings illustrating how the chromatin substrate modulates genome maintenance mechanisms, focusing on the regulatory role of histone variants and post-translational modifications. In particular, we discuss how the pre-existing chromatin landscape impacts DNA damage formation and guides DNA repair pathway choice, and how DNA damage-induced chromatin alterations control DNA damage signaling and repair, and DNA damage segregation through cell divisions. We also highlight that pathological alterations of histone proteins may trigger genome instability by impairing chromosome segregation and DNA repair, thus defining new oncogenic mechanisms and opening up therapeutic options.

Exploring factors influencing choice of DNA double-strand break repair pathways

DNA double-strand breaks (DSBs) represent one of the most severe threats to genomic integrity, demanding intricate repair mechanisms within eukaryotic cells. A diverse array of factors orchestrates the complex choreography of DSB signaling and repair, encompassing repair pathways, such as non-homologous end-joining, homologous recombination, and polymerase-θ-mediated end-joining. This review looks into the intricate decision-making processes guiding eukaryotic cells towards a particular repair pathway, particularly emphasizing the processing of two-ended DSBs. Furthermore, we elucidate the transformative role of Cas9, a site-specific endonuclease, in revolutionizing our comprehension of DNA DSB repair dynamics. Additionally, we explore the burgeoning potential of Cas9's remarkable ability to induce sequence-specific DSBs, offering a promising avenue for precise targeting of tumor cells. Through this comprehensive exploration, we unravel the intricate molecular mechanisms of cellular responses to DSBs, shedding light on both fundamental repair processes and cutting-edge therapeutic strategies.

The impact of developmental stage, tissue type, and sex on DNA double-strand break repair in Drosophila melanogaster.

Accurate repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genome integrity, as failure to repair DSBs can result in cell death. The cell has evolved two main mechanisms for DSB repair: non-homologous end-joining (NHEJ) and homology-directed repair (HDR), which includes single-strand annealing (SSA) and homologous recombination (HR). While certain factors like age and state of the chromatin are known to influence DSB repair pathway choice, the roles of developmental stage, tissue type, and sex have yet to be elucidated in multicellular organisms. To examine the influence of these factors, DSB repair in various embryonic developmental stages, larva, and adult tissues in Drosophila melanogaster was analyzed through molecular analysis of the DR-white assay using Tracking across Indels by DEcomposition (TIDE). The proportion of HR repair was highest in tissues that maintain the canonical (G1/S/G2/M) cell cycle and suppressed in both terminally differentiated and polyploid tissues. To determine the impact of sex on repair pathway choice, repair in different tissues in both males and females was analyzed. When molecularly examining tissues containing mostly somatic cells, males and females demonstrated similar proportions of HR and NHEJ. However, when DSB repair was analyzed in male and female premeiotic germline cells utilizing phenotypic analysis of the DR-white assay, there was a significant decrease in HR in females compared to males. This study describes the impact of development, tissue-specific cycling profile, and, in some cases, sex on DSB repair outcomes, underscoring the complexity of repair in multicellular organisms.