DNA Double-strand Breaks End Resection Research Articles

Effect of PI3-kinase inhibitors on DNA double strand break repair pathways: observations using a site specific DSB induction system.

We established two types of site-specific DNA double strand breaks (DSB) induction systems to elucidate factors which affect the efficiency or quality of DSB repair. For mutation assays, a site specific DSB was generated by the transient expression of a zinc finger nuclease which targets the human HPRT1 gene. A cell line in which time and site specific DSBs can be generated in a HR reporter construct was used for homology-directed repair (HR) analysis. By using these two systems, we investigated the effects of PI3-kinase inhibitors on the efficiency and quality of DSB repair. Ataxia telangiectasia mutated (ATM) kinase inhibition resulted a decrease in mutant frequency and a slight increase in deletion-type mutations accompanied by microhomology. Furthermore, the HR frequency increased significantly when ATM kinase activity was inhibited. Thus, ATM kinase activity might be involved in the suppression of DSB end resection, and this may promote DSB repair through canonical non-homologous end joining.

Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks

Although previous studies have reported that pre-mRNA splicing factors (SFs) are involved in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), their exact role in promoting HR remains poorly understood. Here, we showed that SART1, an SF upregulated in several types of cancer, promotes DSB end resection, an essential first step of HR. The resection-promoting function of SART1 requires phosphorylation at threonine 430 and 695 by ATM/ATR. SART1 is recruited to DSB sites in a manner dependent on transcription and its RS domain. SART1 is epistatic with BRCA1, a major HR factor, in the promotion of resection, especially transcription-associated resection in the G2 phase. SART1 and BRCA1 accumulate at DSB sites in an interdependent manner, and epistatically counteract the resection blockade posed by 53BP1 and RIF1. Furthermore, chromosome analysis demonstrated that SART1 and BRCA1 epistatically suppressed genomic alterations caused by DSB misrepair in the G2 phase. Collectively, these results indicate that SART1 and BRCA1 cooperatively facilitate resection of DSBs arising in transcriptionally active genomic regions in the G2 phase, thereby promoting faithful repair by HR, and suppressing genome instability.

Exacerbated Inflammatory Gene Expression After Impaired G2/M-Checkpoint Arrest in Fibroblasts Derived From a Patient Exhibiting Severe Adverse Effects

Purpose: Recent radiotherapy (RT), such as intensity-modulated radiation therapy and particle RT, has improved the concentration of the radiation field targeting tumors. However, severe adverse effects still occur, possibly due to genetic factors in patients. We aimed to investigate the mechanism of exacerbated inflammation during RT. Methods and MaterialsDermal fibroblasts derived from a patient with severe inflammatory adverse effects during RT were compared with two normal human dermal fibroblasts. Micronuclei formation, G2/M-checkpoint arrest, DNA damage signaling and repair, and inflammatory gene expression were comprehensively examined. Results: We found greater micronuclei formation in radiation-sensitive fibroblasts (RS-Fs) after ionizing radiation (IR). Importantly, RS-Fs exhibited premature G2/M-checkpoint release after IR, which triggers micronuclei formation because RS-Fs undergo mitosis with unrepaired DNA double-strand breaks (DSBs). Additionally, we found that DSB end-resection and activation of the ATR-Chk1 pathway were impaired in RS-Fs after IR. Consistent with the increase in the formation of micronuclei, which can deliver cytosolic nucleic acids resulting in an innate immune response, the expression of genes associated with inflammatory responses was highly upregulated in RS-Fs after IR. Conclusions: Although this is a single case of RT-dependent adverse effect, our findings suggest that impaired G2/M-checkpoint arrest due to the lack of DSB end-resection and activation of the ATR-Chk1 pathway causes exacerbated inflammation during RT; therefore, genes involved in G2/M-checkpoint arrest may be a predictive marker for unexpected inflammatory responses in RT.

Resection of DNA double-strand breaks activates Mre11-Rad50-Nbs1- and Rad9-Hus1-Rad1-dependent mechanisms that redundantly promote ATR checkpoint activation and end processing in Xenopus egg extracts.

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system.

Human HELQ regulates DNA end resection at DNA double-strand breaks and stalled replication forks.

Following a DNA double strand break (DSB), several nucleases and helicases coordinate to generate single-stranded DNA (ssDNA) with 3' free ends, facilitating precise DNA repair by homologous recombination (HR). The same nucleases can act on stalled replication forks, promoting nascent DNA degradation and fork instability. Interestingly, some HR factors, such as CtIP and BRCA1, have opposite regulatory effects on the two processes, promoting end resection at DSB but inhibiting the degradation of nascent DNA on stalled forks. However, the reason why nuclease actions are regulated by different mechanisms in two DNA metabolism is poorly understood. We show that human HELQ acts as a DNA end resection regulator, with opposing activities on DNA end resection at DSBs and on stalled forks as seen for other regulators. Mechanistically, HELQ helicase activity is required for EXO1-mediated DSB end resection, while ssDNA-binding capacity of HELQ is required for its recruitment to stalled forks, facilitating fork protection and preventing chromosome aberrations caused by replication stress. Here, HELQ synergizes with CtIP but not BRCA1 or BRCA2 to protect stalled forks. These findings reveal an unanticipated role of HELQ in regulating DNA end resection at DSB and stalled forks, which is important for maintaining genome stability.

Robust Radiosensitization by Combined Treatment of Cancer Cells with Talazoparib and Polθ Inhibitors

The PARP inhibitor talazoparib is synthetically lethal with HR-defective tumors and functions as a potent radiosensitizer specifically of cancer cells. Talazoparib exerts this unique radiosensitizing property by shifting ionizing radiation (IR)-induced DNA double strand break (DSB) repair towards error-prone alternative end-joining (alt-EJ). DNA polymerase theta (Polθ, encoded by POLQ) is a key component of alt-EJ. Here, we tested the hypothesis that inhibition of alt-EJ with Polθ ablation or using specific small molecule inhibitors can further increase talazoparib-induced radiosensitization. U2OS, A549, and their corresponding POLQ ablated/mutant cell lines were treated with talazoparib and/or Polθ inhibitors ART558/novobiocin prior to irradiation. siRNAs against CtIP, MRE11, EXO1; and a specific inhibitor of DNA2 were employed to suppress DNA end resection. Radiosensitization was assessed by clonogenic survival. Olaparib, rucaparib, and veliparib were also tested under similar conditions. DSB repair and end resection were measured by scoring γH2AX and RPA nuclear foci, respectively. Chromosomal abnormalities were assessed using G2-specific cytogenetics analysis. Genetic ablation or pharmacological inhibition of Polθ robustly enhanced talazoparib mediated radiosensitization by ∼40-70%. Notably, Polθ inhibition had a much lower effect (by ∼7-17%) when combined with other clinically used PARP inhibitors, olaparib, rucaparib, and veliparib. Polθ inhibition significantly suppressed talazoparib-induced translocation formation in irradiated cells. In addition, combined treatment with Polθ inhibitor and talazoparib attenuated DSB repair, resulting in ∼60% unresolved γH2AX foci and ∼40% unresolved chromatid breaks at 5h post IR. Talazoparib promoted resection of DNA ends as demonstrated by an increase in RPA foci. The resection process requires the activities of CtIP and MRE11, but not of DNA2 or EXO1. Finally, CtIP and MRE11 knockdown impaired radiosensitization following a combined talazoparib/Polθ inhibition treatment. Talazoparib increases the reliance of irradiated cancer cells on Polθ-mediated alt-EJ owing to the increased CtIP/MRE11-dependent resection it produces. Combining talazoparib with Polθ inhibitors has therefore great potential in improving radiotherapy of human tumors.

CCAR2 functions downstream of the Shieldin complex to promote double-strand break end-joining

The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.

BMN673 Is a PARP Inhibitor with Unique Radiosensitizing Properties: Mechanisms and Potential in Radiation Therapy.

BMN673 is a relatively new PARP inhibitor (PARPi) that exhibits superior efficacy in vitro compared to olaparib and other clinically relevant PARPi. BMN673, similar to most clinical PARPi, inhibits the catalytic activities of PARP-1 and PARP-2 and shows impressive anticancer potential as monotherapy in several pre-clinical and clinical studies. Tumor resistance to PARPi poses a significant challenge in the clinic. Thus, combining PARPi with other treatment modalities, such as radiotherapy (RT), is being actively pursued to overcome such resistance. However, the modest to intermediate radiosensitization exerted by olaparib, rucaparib, and veliparib, limits the rationale and the scope of such combinations. The recently reported strong radiosensitizing potential of BMN673 forecasts a paradigm shift on this front. Evidence accumulates that BMN673 may radiosensitize via unique mechanisms causing profound shifts in the balance among DNA double-strand break (DSB) repair pathways. According to one of the emerging models, BMN673 strongly inhibits classical non-homologous end-joining (c-NHEJ) and increases reciprocally and profoundly DSB end-resection, enhancing error-prone DSB processing that robustly potentiates cell killing. In this review, we outline and summarize the work that helped to formulate this model of BMN673 action on DSB repair, analyze the causes of radiosensitization and discuss its potential as a radiosensitizer in the clinic. Finally, we highlight strategies for combining BMN673 with other inhibitors of DNA damage response for further improvements.

O-GlcNAc transferase is important for homology-directed repair

O-Linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) to serine or threonine residues is a reversible and dynamic post-translational modification. O-GlcNAc transferase (OGT) is the only enzyme for O-GlcNAcylation, and is a potential cancer therapeutic target in combination with clastogenic (i.e., chromosomal breaking) therapeutics. Thus, we sought to examine the influence of O-GlcNAcylation on chromosomal break repair. Using a set of DNA double strand break (DSB) reporter assays, we found that the depletion of OGT, and its inhibition with a small molecule each caused a reduction in repair pathways that involve use of homology: RAD51-dependent homology-directed repair (HDR), and single strand annealing. In contrast, such OGT disruption did not obviously affect chromosomal break end joining, and furthermore caused an increase in homology-directed gene targeting. Such disruption in OGT also caused a reduction in clonogenic survival, as well as modifications to cell cycle profiles, particularly an increase in G1-phase cells. We also examined intermediate steps of HDR, finding no obvious effects on an assay for DSB end resection, nor for RAD51 recruitment into ionizing radiation induced foci (IRIF) in proliferating cells. However, we also found that the influence of OGT on HDR and homology-directed gene targeting were dependent on RAD52, and that OGT is important for RAD52 IRIF in proliferating cells. Thus, we suggest that OGT is important for regulation of HDR that is partially linked to RAD52 function.

Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers.

Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Abstract 796: Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers

Abstract DNA repair pathway inhibitors are a new class of anti-cancer drugs that are advancing in clinical trials. While inhibitors of targets in the Non-Homologous End Joining (NHEJ) DNA repair pathway, such as DNA-dependent protein kinase (DNA-PK), are available for clinical use, it remains unclear which cancers are vulnerable to these agents. In a genome-wide CRISPR knockout screen with the DNA-PK inhibitor M3814, we identify loss of POLQ, encoding polymerase theta, and other genes in the microhomology-mediated end-joining (MMEJ) pathway as key predictors of sensitivity to DNA-PK inhibition, whereas loss of TP53 conferred resistance to DNA-PK inhibition. Inhibition of DNA-PK led to increased DNA double strand break end-resection, increased expression of polymerase theta, and activation of MMEJ repair. Combined DNA-PK inhibition by M3814 and polymerase theta inhibition by novobiocin resulted in synthetic lethality mediated by the accumulation of resected DNA and apoptosis. Significantly, this drug combination efficiently killed TP53-deficient human patient-derived xenografts and the corresponding tumor organoids. Taken together, our results provide a rationale for the combination of an inhibitor of DNA-PK mediated NHEJ and an inhibitor of polymerase theta mediated MMEJ in an anti-cancer trial. If DNA-PK is inhibited, cancers develop a hyper-dependence on MMEJ and an upregulation of DNA double strand break end resection and polymerase theta expression. Similarly, P53-deficiency which confers resistance to DNA-PK inhibition, also leads to a hyper-dependence on MMEJ and an upregulation of polymerase theta expression. Thus, a combination of DNA-PK and polymerase theta inhibitors may provide a precision treatment strategy for TP53-mutant solid tumors, known to account for 50% of newly diagnosed cancers. Citation Format: Jeffrey Patterson-Fortin, Arindam Bose, Wei-Chih Tsai, Carter Grochala, Huy Nguyen, Jia Zhou, Kalindi Parmar, Jean-Bernard Lazaro, Joyce Liu, Kelsey McQueen, Geoffrey I. Shapiro, David Kozono, Alan D. D'Andrea. Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 796.

MRNIP condensates promote DNA double-strand break sensing and end resection

The rapid recognition of DNA double-strand breaks (DSBs) by the MRE11/RAD50/NBS1 (MRN) complex is critical for the initiation of DNA damage response and DSB end resection. Here, we show that MRN complex interacting protein (MRNIP) forms liquid-like condensates to promote homologous recombination-mediated DSB repair. The intrinsically disordered region is essential for MRNIP condensate formation. Mechanically, the MRN complex is compartmentalized and concentrated into MRNIP condensates in the nucleus. After DSB formation, MRNIP condensates move to the damaged DNA rapidly to accelerate the binding of DSB by the concentrated MRN complex, therefore inducing the autophosphorylation of ATM and subsequent activation of DNA damage response signaling. Meanwhile, MRNIP condensates-enhanced MRN complex loading further promotes DSB end resection. In addition, data from xenograft models and clinical samples confirm a correlation between MRNIP and radioresistance. Together, these results reveal an important role of MRNIP phase separation in DSB response and the MRN complex-mediated DSB end resection.

Modulating DNA Repair Pathways to Diversify Genomic Alterations in Saccharomyces cerevisiae.

ABSTRACTNuclease based genome editing systems have emerged as powerful tools to drive genomic alterations and enhance genome evolution via precise engineering in the various human and microbial cells. However, error-prone DNA repair has not been well studied previously to generate diverse genomic alterations and novel phenotypes. Here, we systematically investigated the potential interplay between DNA double strand break (DSB) repair and genome editing tools, and found that modulating the DSB end resection proteins could significantly improve mutational efficiency and diversity without exogenous DNA template in yeast. Deleting SAE2, EXO1, or FUN30, or overexpressing MRE11-H125N (nuclease-dead allele of MRE11), for DSB end resection markedly increased the efficiency of CRISPR/SpCas9 (more than 22-fold) and CRISPR/AsCpf1 (more than 30-fold)-induced mutagenesis. Deleting SAE2 or overexpressing MRE11-H125N substantially diversified CRISPR/SpCas9 or AsCpf1-induced mutation 2–3-fold at URA3 locus, and 3–5-fold at ADE2 locus. Thus, the error-prone DNA repair protein was employed to develop a novel mutagenic genome editing (mGE) strategy, which can increase the mutation numbers and effectively improve the ethanol/glycerol ratio of Saccharomyces cerevisiae through modulating the expression of FPS1 and GPD1. This study highlighted the feasibility of potentially reshaping the capability of genome editing by regulating the different DSB repair proteins and can thus expand the application of genome editing in diversifying gene expression and enhancing genome evolution.IMPORTANCE Most of the published papers about nuclease-assisted genome editing focused on precision engineering in human cells. However, the topic of inducing mutagenesis via error-prone repair has often been ignored in yeast. In this study, we reported that perturbing DNA repair, especially modifications of the various DSB end resection-related proteins, could greatly improve the mutational efficiency and diversity, and thus functionally reshape the capability of the different genome editing tools without requiring an exogenous DNA template in yeast. Specifically, mutagenic genome editing (mGE) was developed based on CRISPR/AsCpf1 and MRE11-H125N overexpression, and used to generate promoters of different strengths more efficiently. Thus, this work provides a novel method to diversify gene expression and enhance genome evolution.

Roles of the SUMO-related enzymes, PIAS1, PIAS4, and RNF4, in DNA double-strand break repair by homologous recombination

Post-translational modification of proteins by small ubiquitin-like modifier (SUMO) is known to be involved in a variety of cellular events. This modification, called SUMOylation, is carried out by the E1 activating enzyme, the E2 conjugating enzyme, and multiple E3 ligases. Previous studies have demonstrated that the SUMO E3 ligases, protein inhibitors of activated STAT 1 (PIAS1) and 4 (PIAS4), and the SUMO-targeted ubiquitin ligase, RING finger protein 4 (RNF4), play important roles in the repair of DNA double-strand breaks (DSBs). However, the mechanism by which these SUMO-related enzymes promote DSB repair is still poorly understood. In the present study, we focused on homologous recombination (HR), the most accurate DSB repair pathway, and aimed to elucidate the mechanism by which PIAS1, PIAS4, and RNF4 promote HR. In γ-ray-irradiated normal human fibroblasts, DSB end resection and RAD51 loading, the two essential steps of HR, were significantly impaired by small interfering RNA (siRNA)-mediated depletion of PIAS1, PIAS4, or RNF4. The recruitment of BRCA1, a major HR factor, to DSB sites was reduced in cells depleted of these SUMO-related enzymes. Consistent with the role of BRCA1 in counteracting the p53-binding protein 1 (53BP1)-mediated resection blockade, 53BP1 depletion rescued the reduced resection and RAD51 loading in the cells depleted of PIAS1, PIAS4, or RNF4. Moreover, Rap1-interacting factor 1 (RIF1), a resection inhibitor downstream of 53BP1, became more abundant at DSBs when PIAS1, PIAS4, RNF4, or BRCA1 was depleted. Importantly, the concomitant depletion of BRCA1 with either one of the SUMO-related enzymes did not further increase RIF1 at DSBs, when compared to single depletion of BRCA1. Collectively, these results suggest that PIAS1, PIAS4, RNF4, and BRCA1 work epistatically to counteract 53BP1/RIF1-mediated resection blockade, thereby promoting resection.

Interaction of Wwox with Brca1 and associated complex proteins prevents premature resection at double-strand breaks and aberrant homologous recombination

Down regulation of Wwox protein expression occurs in many cancers, contributing to insensitivity to ionizing radiation (IR) and platinum drug treatments. Patients with reduced Wwox expression in their cancer tissue show decreased overall survival following these treatments, in accord with our earlier finding that reduced Wwox protein expression in cancers is associated with changes in choice of DNA double-strand break (DSB) repair pathway. Our current investigation of mechanisms underlying the initial choice of repair by homologous recombination/single-strand annealing (HR/SSA) in Wwox-deficient cells, showed immediate DNA end-resection at DSBs following IR, abrogating initial repair by the expected non-homologous end-joining (NHEJ) pathway. Mechanisms supporting the expected choice of DSB repair by NHEJ in Wwox-sufficient cells are: 1) direct recruitment of Wwox protein binding to Brca1 through the Brca1 981PPLF984 Wwox-binding motif; 2) possible Wwox blocking of Brca1-Rad50 interaction and of Brca1 activation by Chk2 phosphorylation of Brca1 S988; 3) Wwox suppression of Brca1 interaction with the B and C complex proteins, Brip1 and CtIP, thereby delaying the process of DSB end-resection post-IR. Wwox deficiency, instead, leads to early formation of the Brca1-CtIP/MRN complex at induced DSBs, stimulating immediate post-IR end-resection. This premature resection at DNA DSBs leads to inappropriate HR/SSA repair not restricted to late S/G2 cell cycle phases, and increases mutations in genomes of radiation or platinum-resistant colonies. Prevention of premature initiation of end-resection, by combining Chk2 inhibition with IR or carboplatin treatment, successfully sensitized IR and platinum-resistant Wwox-deficient cells by synthetic lethality, but did not alter response of Wwox-sufficient cells. Our results establish Wwox as a biomarker for treatment response and provide potential targets, such as Chk2, for reversal of treatment resistance.

RB Regulates DNA Double Strand Break Repair Pathway Choice by Mediating CtIP Dependent End Resection.

Inactivation of the retinoblastoma tumor suppressor gene (RB1) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.

FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku

The balance between major DNA double-strand break (DSB) repair pathways is influenced by binding of the Ku complex, a XRCC5/6 heterodimer, to DSB ends, initiating non-homologous end joining (NHEJ) but preventing additional DSB end resection and homologous recombination (HR). However, the key molecular cue for Ku recruitment to DSB sites is unknown. Here, we report that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku. Upon DSB induction, SIRT1 translocates to the nucleus and deacetylates FOXL2 at lysine 124, leading to liberation of XRCC5 and XRCC6 from FOXL2 and formation of the Ku complex. FOXL2 ablation enhances Ku recruitment to DSB sites, imbalances DSB repair kinetics by accelerating NHEJ and inhibiting HR, and thus leads to catastrophic genomic events. Our study unveils the SIRT1-(de)acetylated FOXL2-Ku axis that governs the balance of DSB repair pathways to maintain genome integrity.

BLM has Contrary Effects on Repeat-Mediated Deletions, based on the Distance of DNA DSBs to a Repeat and Repeat Divergence

SUMMARYRepeat-mediated deletions (RMDs) often involve repetitive elements (e.g., short interspersed elements) with sequence divergence that is separated by several kilobase pairs (kbps). We have examined RMDs induced by DNA double-strand breaks (DSBs) under varying conditions of repeat sequence divergence (identical versus 1% and 3% divergent) and DSB/repeat distance (16 bp–28.4 kbp). We find that the BLM helicase promotes RMDs with long DSB/repeat distances (e.g., 28.4 kbp), which is consistent with a role in extensive DSB end resection, because the resection nucleases EXO1 and DNA2 affect RMDs similarly to BLM. In contrast, BLM suppresses RMDs with sequence divergence and intermediate (e.g., 3.3 kbp) DSB/repeat distances, which supports a role in heteroduplex rejection. The role of BLM in heteroduplex rejection is not epistatic with MSH2 and is independent of the annealing factor RAD52. Accordingly, the role of BLM on RMDs is substantially affected by DSB/repeat distance and repeat sequence divergence.

Hypersensitivity to DNA double-strand breaks associated with PARG deficiency is suppressed by exo-1 and polq-1 mutations in Caenorhabditiselegans.

Deficiency of either of the two homologs of poly(ADP-ribose) glycohydrolase (PARG), PARG-1 and PARG-2, in Caenorhabditiselegans leads to hypersensitivity to ionizing radiation (IR). In the germ cells of parg-2 mutant worms, the dissipation of recombinase RAD-51 foci was slower than in wild-type (WT) cells, suggesting defects in DNA double-strand break (DSB) repair via homologous recombination (HR). Nevertheless, RPA-1, the large subunit of replication protein A, accumulated faster in parg-2 worms and disappeared earlier than in WT worms. This accelerated RPA-1 accumulation may result from the enhanced expression of exonuclease-1 (EXO-1) after IR treatment. Accordingly, an exo-1 mutation reduced IR sensitivity and accumulation of RPA-1 in parg-2 worms. A mutation of polq-1, encoding for a key factor in the alternative end-joining (Alt-EJ) pathway, suppressed the IR hypersensitivity phenotype of parg-2 worms and normalized the kinetics of RAD-51 dissipation. This indicates that error-prone Alt-EJ may mediate DSB repair in parg-2 worms, causing hypersensitivity to IR. In summary, PARG-2 deficiency in C.elegans causes hyperactive DSB end resection likely through EXO-1 overproduction. DSBs with long single-stranded DNA ends in parg-2 worms are thought to be repaired by Alt-EJ instead of HR, causing genomic instability.

RBX1 prompts degradation of EXO1 to limit the homologous recombination pathway of DNA double-strand break repair in G1 phase

End resection of DNA double-strand breaks (DSBs) to form 3′ single-strand DNA (ssDNA) is critical to initiate the homologous recombination (HR) pathway of DSB repair. HR pathway is strictly limited in the G1-phase cells because of lack of homologous DNA as the templates. Exonuclease 1 (EXO1) is the key molecule responsible for 3′ ssDNA formation of DSB end resection. We revealed that EXO1 is inactivated in G1-phase cells via ubiquitination-mediated degradation, resulting from an elevated expression level of RING-box protein 1 (RBX1) in G1 phase. The increased RBX1 significantly prompted the neddylation of Cullin1 and contributed to the G1 phase-specific degradation of EXO1. Knockdown of RBX1 remarkedly attenuated the degradation of EXO1 and increased the end resection and HR activity in γ-irradiated G1-phase cells, as demonstrated by the increased formation of RPA32, BrdU, and RAD51 foci. And EXO1 depletion mitigated DNA repair defects due to RBX1 reduction. Moreover, increased autophosphorylation of DNA-PKcs at S2056 was found to be responsible for the higher expression level of the RBX1 in the G1 phase. Inactivation of DNA-PKcs decreased RBX1 expression, and simultaneously increased EXO1 expression and DSB end resection in G1-phase cells. This study demonstrates a new mechanism for restraining the HR pathway of DNA DSB repair in G1 phase via RBX1-prompted inactivation of EXO1.