Mre11 Nuclease Research Articles

The MRE11-RAD50-NBS1 complex both starts and extends DNA end resection in mouse meiosis.

Nucleolytic resection of DNA ends is critical for homologous recombination, but its mechanism is not fully understood, particularly in mammalian meiosis. Here we examine roles of the conserved MRN complex (MRE11, RAD50, and NBS1) through genome-wide analysis of meiotic resection in mice with various MRN mutations, including several that cause chromosomal instability in humans. Meiotic DSBs form at elevated levels but remain unresected if Mre11 is conditionally deleted, thus MRN is required for both resection initiation and regulation of DSB numbers. Resection lengths are reduced to varying degrees in MRN hypomorphs or if MRE11 nuclease activity is attenuated in a conditional nuclease-dead Mre11 model. These findings unexpectedly establish that MRN is needed for longer-range extension of resection, not just resection initiation. Finally, resection defects are additively worsened by combining MRN and Exo1 mutations, and mice that are unable to initiate resection or have greatly curtailed resection lengths experience catastrophic spermatogenic failure. Our results elucidate multiple functions of MRN in meiotic recombination, uncover unanticipated relationships between short- and long-range resection, and establish the importance of resection for mammalian meiosis.

"Bridging the DNA divide": Understanding the interplay between replication- gaps and homologous recombination proteins RAD51 and BRCA1/2

A key but often neglected component of genomic instability is the emergence of single-stranded DNA (ssDNA) gaps during DNA replication in the absence of functional homologous recombination (HR) proteins, such as RAD51 and BRCA1/2. Research in prokaryotes has shed light on the dual role of RAD51's bacterial ortholog, RecA, in HR and the protection of replication forks, emphasizing its essential role in preventing the formation of ssDNA gaps, which is vital for cellular viability. This phenomenon was corroborated in eukaryotic cells deficient in HR, where the formation of ssDNA gaps within newly synthesized DNA and their subsequent processing by the MRE11 nuclease were observed. Without functional HR proteins, cells employ alternative ssDNA gap-filling mechanisms to ensure survival, though this compensatory response can compromise genomic stability. A notable example is the involvement of the translesion synthesis (TLS) polymerase POLζ, along with the repair protein POLθ, in the suppression of replicative ssDNA gaps. Persistent ssDNA gaps may result in replication fork collapse, chromosomal anomalies, and cell death, which contribute to cancer progression and resistance to therapy. Elucidating the processes that avert ssDNA gaps and safeguard replication forks is critical for enhancing cancer treatment approaches by exploiting the vulnerabilities of cancer cells in these pathways

MRE11 and TREX1 control senescence by coordinating replication stress and interferon signaling

Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RASV12 oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RASV12. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-β was sufficient to induce RS and DNA damage, independent of RASV12 induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.

Binding of the TRF2 iDDR motif to RAD50 highlights a convergent evolutionary strategy to inactivate MRN at telomeres.

Telomeres protect chromosome ends from unscheduled DNA repair, including from the MRN (MRE11, RAD50, NBS1) complex, which processes double-stranded DNA breaks (DSBs) via activation of the ATM kinase, promotes DNA end-tethering aiding the non-homologous end-joining (NHEJ) pathway, and initiates DSB resection through the MRE11 nuclease. A protein motif (MIN, for MRN inhibitor)inhibits MRN at budding yeast telomeres by binding to RAD50 andevolved at least twice, in unrelated telomeric proteins Rif2 and Taz1. We identify the iDDR motif of human shelterin protein TRF2 as a third example of convergent evolution for thistelomericmechanism for binding MRN, despite the iDDR lacking sequence homology to the MIN motif. CtIP is required for activation of MRE11 nuclease action, and we provide evidence for binding of a short C-terminal region of CtIP to a RAD50 interface that partly overlaps with the iDDR binding site, indicating that the interaction is mutually exclusive. In addition, we show that the iDDR impairs the DNA binding activity of RAD50. These results highlight direct inhibition of MRN action as a crucial role of telomeric proteins across organisms and point to multiple mechanisms enforced by the iDDR to disable the many activities of the MRN complex.

Molecular insights into the activation of Mre11-Rad50 endonuclease activity by Sae2/CtIP

In Saccharomyces cerevisiae (S.cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX invitro and invivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.

UFL1 triggers replication fork degradation by MRE11 in BRCA1/2-deficient cells.

The stabilization of stalled forks has emerged as a crucial mechanism driving resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient tumors. Here, we identify UFL1, a UFM1-specific E3 ligase, as a pivotal regulator of fork stability and the response to PARP inhibitors in BRCA1/2-deficient cells. On replication stress, UFL1 localizes to stalled forks and catalyzes the UFMylation of PTIP, a component of the MLL3/4 methyltransferase complex, specifically at lysine 148. This modification facilitates the assembly of the PTIP-MLL3/4 complex, resulting in the enrichment of H3K4me1 and H3K4me3 at stalled forks and subsequent recruitment of the MRE11 nuclease. Consequently, loss of UFL1, disruption of PTIP UFMylation or overexpression of the UFM1 protease UFSP2 protects nascent DNA strands from extensive degradation and confers resistance to PARP inhibitors in BRCA1/2-deficient cells. These findings provide mechanistic insights into the processes underlying fork instability in BRCA1/2-deficient cells and offer potential therapeutic avenues for the treatment of BRCA1/2-deficient tumors.

APLF facilitates interstrand DNA crosslink repair and replication fork protection to confer cisplatin resistance.

Replication stress converts the stalled forks into reversed forks, which is an important protection mechanism to prevent fork degradation and collapse into poisonous DNA double-strand breaks (DSBs). Paradoxically, the mechanism also acts in cancer cells to contribute to chemoresistance against various DNA-damaging agents. PARP1 binds to and is activated by stalled forks to facilitate fork reversal. Aprataxin and polynucleotide kinase/phosphatase-like factor (APLF) binds to PARP1 through the poly(ADP-ribose) zinc finger (PBZ) domain and is known to be involved in non-homologous end joining (NHEJ). Here, we identify a novel function of APLF involved in interstrand DNA crosslink (ICL) repair and fork protection. We demonstrate that PARP1 activity facilitates the APLF recruitment to stalled forks, enabling the FANCD2 recruitment to stalled forks. The depletion of APLF sensitizes cells to cisplatin, impairs ICL repair, reduces the FANCD2 recruitment to stalled forks, and results in nascent DNA degradation by MRE11 nucleases. Additionally, cisplatin-resistant cancer cells show high levels of APLF and homologous recombination-related gene expression. The depletion of APLF sensitizes cells to cisplatin and results in fork instability. Our results reveal the novel function of APLF to facilitate ICL repair and fork protection, thereby contributing to cisplatin-resistant phenotypes of cancer cells.

Abstract 2690: The role of PCNA tyrosine phosphorylation in evading innate immune surveillance

Abstract Cancer cells are continually exposed to intrinsic and extrinsic proliferative stresses that can send the genome stability awry. The aberrant DNA metabolism, evolving through deregulated replicative growth in cancer cells, is increasingly recognized as a major mechanism bridging the crosstalk between the immune microenvironment and tumor tissue, thereby either promoting or suppressing tumor progression. However, the triggering and regulation of these mechanisms remain largely unknown. Proliferating cell nuclear antigen (PCNA) and its homologs are the evolutionarily conserved central components of the DNA replication machinery. In eukaryotic cells, PCNA forms homotrimeric rings encircling the DNA double helix to coordinate the complex processes of DNA replication and damage repair, directed at least in part by differential post-translational modifications of the PCNA protein. In cancer cells, growth signal-stimulated phosphorylation at tyrosine 211 of PCNA (pY211-PCNA) is known to play an important function for active proliferation. Here, we demonstrate that inhibition of pY211 disrupts the processivity of replication forks. This disruption triggers single-stranded DNA (ssDNA) production catalyzed by the MRE11 nuclease, activating the cGAS-STING axis to launch an anti-tumor immune response by natural killer (NK) cells. We further show that the pY211 regulates site-specific post-translational modifications of MRE11, and loss of pY211 promotes the endonuclease mode of MRE11. This causes ssDNA generation in the cytosol, subsequently inducing type I interferon-activated cytotoxicity by NK cells and fostering an anti-tumor immunity that abrogates distant metastasis. Mechanistically, the pY211 determines the recruitment of the writer or eraser of the modification on MRE11, thus tuning the nucleolytic modes of MRE11 and, consequently, ssDNA generation. Therapeutic measures promoting MRE11-dependent ssDNA enhance the response of triple-negative breast cancer to cytotoxic killing mediated by endogenous or allogenic NK cells in mice. In breast cancer patients, the level of cytosolic ssDNA is inversely correlated with the expression of the eraser and pY211-PCNA, which is associated with poor overall survival in cancer patients. Our studies shed new light on the shaping of the tumor immune microenvironment and demonstrate the potential to develop therapeutic approaches that proactively leverage genomic instability and immune activation to target malignant tumors. Citation Format: Chuan-Chun Lee, Wan-Rong Wu, Yi-Chun Shen, Feng-Chi Chung, Yuan-Liang Wang, You-Zhe Lin, Chih-Hao Lu, Wei-Chung Cheng, Han Chang, Liang-Chih Liu, Ji-An Liang, Chang- Fang Chiu, Mien-Chie Hung, Shao-Chun Wang. The role of PCNA tyrosine phosphorylation in evading innate immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2690.

Abstract IA014: BRCA2 promotes genomic integrity and therapy resistance primarily through its role in homology-directed repair

Abstract Tumor suppressor BRCA2 functions in multiple pathways. The role of BRCA2 in homologous recombination, also termed homology-directed repair (HDR), is particularly well known. We also showed that it has a key role in the protection of stalled replication forks from the MRE11 nuclease. Moreover, BRCA2 also suppresses the formation of single-stranded gaps during replication. We have attempted to delineate the relative contributions of these three pathways to the maintenance of genome integrity and chemotherapy response using separation of function alleles. First, we report that mouse and human cells require a RAD51 filament stabilization motif in BRCA2 for both fork protection and gap suppression but not HDR. In mice, loss of fork protection and gap suppression do not compromise genome instability or shorten tumor latency. By contrast, HDR deficiency increases spontaneous and replication stress-induced chromosome aberrations and tumor predisposition. Unlike with HDR, fork protection and gap suppression defects are also observed in Brca2 heterozygous cells from mice, likely due to reduced RAD51 stabilization at stalled forks and gaps. Sensitivity to the PARP inhibitor olaparib is greatest for HDR-deficient cells, although fork protection/gap suppression-defective cells show a weak sensitivity. Gaps arise from PRIMPOL activity, which is associated with sensitivity to 5-hydroxymethyl-2’-deoxyuridine (hmdU) and DNPH1 inhibition due to the formation of SMUG1-generated abasic sites and is exacerbated by poly(ADP-ribose) polymerase inhibition. In summary, this work demonstrates that HDR deficiency has the major role in generating sensitivity to chemotherapeutics, although gap suppression deficiency may be targeted by specific agents. Citation Format: Pei Xin Lim, Maria Jasin. BRCA2 promotes genomic integrity and therapy resistance primarily through its role in homology-directed repair [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA014.

USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

DNA replication stress-induced fork arrest represents a significant threat to genomic integrity. One major mechanism of replication restart involves repriming downstream of the arrested fork by PRIMPOL, leaving behind a single-stranded DNA (ssDNA) gap. Accumulation of nascent strand ssDNA gaps has emerged as a possible determinant of the cellular hypersensitivity to genotoxic agents in certain genetic backgrounds such as BRCA deficiency, but how gaps are converted into cytotoxic structures is still unclear. Here, we investigate the processing of PRIMPOL-dependent ssDNA gaps upon replication stress induced by hydroxyurea and cisplatin. We show that gaps generated in PRIMPOL-overexpressing cells are expanded in the 3'-5' direction by the MRE11 exonuclease, and in the 5'-3' direction by the EXO1 exonuclease. This bidirectional exonucleolytic gap expansion ultimately promotes their conversion into DSBs. We moreover identify the de-ubiquitinating enzyme USP1 as a critical regulator of PRIMPOL-generated ssDNA gaps. USP1 promotes gap accumulation during S-phase, and their expansion by the MRE11 and EXO1 nucleases. This activity of USP1 is linked to its role in de-ubiquitinating PCNA, suggesting that PCNA ubiquitination prevents gap accumulation during replication. Finally, we show that USP1 depletion suppresses DSB formation in PRIMPOL-overexpressing cells, highlighting an unexpected role for USP1 in promoting genomic instability under these conditions.

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding.

The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.

Response of Fibroblasts from Menkes' and Wilson's Copper Metabolism-Related Disorders to Ionizing Radiation: Influence of the Nucleo-Shuttling of the ATM Protein Kinase.

Menkes' disease (MD) and Wilson's disease (WD) are two major copper (Cu) metabolism-related disorders caused by mutations of the ATP7A and ATP7B ATPase gene, respectively. While Cu is involved in DNA strand breaks signaling and repair, the response of cells from both diseases to ionizing radiation, a common DNA strand breaks inducer, has not been investigated yet. To this aim, three MD and two WD skin fibroblasts lines were irradiated at two Gy X-rays and clonogenic cell survival, micronuclei, anti-γH2AX, -pATM, and -MRE11 immunofluorescence assays were applied to evaluate the DNA double-strand breaks (DSB) recognition and repair. MD and WD cells appeared moderately radiosensitive with a delay in the radiation-induced ATM nucleo-shuttling (RIANS) associated with impairments in the DSB recognition. Such delayed RIANS was notably caused in both MD and WD cells by a highly expressed ATP7B protein that forms complexes with ATM monomers in cytoplasm. Interestingly, a Cu pre-treatment of cells may influence the activity of the MRE11 nuclease and modulate the radiobiological phenotype. Lastly, some high-passage MD cells cultured in routine may transform spontaneously becoming immortalized. Altogether, our findings suggest that exposure to ionizing radiation may impact on clinical features of MD and WD, which requires cautiousness when affected patients are submitted to radiodiagnosis and, eventually, radiotherapy.

Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting

A synthetic lethal relationship exists between disruption of polymerase theta (Polθ), and loss of either 53BP1 or homologous recombination (HR) proteins, including BRCA1; however, the mechanistic basis of these observations are unclear. Here we reveal two distinct mechanisms of Polθ synthetic lethality, identifying dual influences of 1) whether Polθ is lost or inhibited, and 2) the underlying susceptible genotype. Firstly, we find that the sensitivity of BRCA1/2- and 53BP1-deficient cells to Polθ loss, and 53BP1-deficient cells to Polθ inhibition (ART558) requires RAD52, and appropriate reduction of RAD52 can ameliorate these phenotypes. We show that in the absence of Polθ, RAD52 accumulations suppress ssDNA gap-filling in G2/M and encourage MRE11 nuclease accumulation. In contrast, the survival of BRCA1-deficient cells treated with Polθ inhibitor are not restored by RAD52 suppression, and ssDNA gap-filling is prevented by the chemically inhibited polymerase itself. These data define an additional role for Polθ, reveal the mechanism underlying synthetic lethality between 53BP1, BRCA1/2 and Polθ loss, and indicate genotype-dependent Polθ inhibitor mechanisms.

Acetyl Transferase EP300 Deficiency Leads to Chronic Replication Stress in Adult T-Cell Leukemia/Lymphoma

Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a disease model to study EP300 deficiency and an EP300-selective PROTAC degrader, as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, transmission of unrepaired inherited DNA lesions in the subsequent G1-phase and accumulation of cytosolic DNA in EP300-deficient cells. However, EP300 deficient cells show a significant defect in innate immune system activation. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress, persistent genomic instability and innate immune system evasion underlie aggressive chemo-resistant tumorigenesis in humans.

Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ-dependent mechanism

Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease activity. Foldback priming at DNA double-strand breaks (DSBs) is one mechanism proposed for the generation of inverted duplications. However, the other pathway steps have not been fully elucidated. Here, we show that a DSB induced near natural inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase δ proof-reading activity, but not Rad1 nuclease, trims the heterologous flaps formed after foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol δ catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric inversion chromosome. Finally, we show that stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences or by deletion of one centromere.

Multi-step processing of replication stress-derived nascent strand DNA gaps by MRE11 and EXO1 nucleases

Accumulation of single stranded DNA (ssDNA) gaps in the nascent strand during DNA replication has been associated with cytotoxicity and hypersensitivity to genotoxic stress, particularly upon inactivation of the BRCA tumor suppressor pathway. However, how ssDNA gaps contribute to genotoxicity is not well understood. Here, we describe a multi-step nucleolytic processing of replication stress-induced ssDNA gaps which converts them into cytotoxic double stranded DNA breaks (DSBs). We show that ssDNA gaps are extended bidirectionally by MRE11 in the 3’−5’ direction and by EXO1 in the 5’−3’ direction, in a process which is suppressed by the BRCA pathway. Subsequently, the parental strand at the ssDNA gap is cleaved by the MRE11 endonuclease generating a double strand break. We also show that exposure to bisphenol A (BPA) and diethylhexyl phthalate (DEHP), which are widespread environmental contaminants due to their use in plastics manufacturing, causes nascent strand ssDNA gaps during replication. These gaps are processed through the same mechanism described above to generate DSBs. Our work sheds light on both the relevance of ssDNA gaps as major determinants of genomic instability, as well as the mechanism through which they are processed to generate genomic instability and cytotoxicity.

The ribonuclease domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response.

The SLFN11 gene participates in cell fate decision following cancer chemotherapy and encodes the N-terminal ribonuclease (RNase) domain and the C-terminal helicase/ATPase domain. How these domains contribute to the chemotherapeutic response remains controversial. Here, we expressed SLFN11 containing mutations in two critical residues required for RNase activity in SLFN11-/- cells. We found that this mutant was still able to suppress DNA damage tolerance, destabilized the stalled replication forks, and perturbed recruitment of the fork protector RAD51. In contrast, we confirmed that the helicase domain was essential to accelerate fork degradation. The fork degradation by the RNase mutant was dependent on both DNA2 and MRE11 nuclease, but not on MRE11's novel interactor FXR1. Collectively, these results supported the view that the RNase domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response.

Selective Killing of BRCA2-Deficient Ovarian Cancer Cells via MRE11 Blockade

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/− cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions

The DNA double-strand break repair complex Mre11-Rad50-Nbs1 (MRN) detects and nucleolytically processes DNA ends, activates the ATM kinase, and tethers DNA at break sites. How MRN can act both as nuclease and scaffold protein is not well understood. The cryo-EM structure of MRN from Chaetomium thermophilum reveals a 2:2:1 complex with a single Nbs1 wrapping around the autoinhibited Mre11 nuclease dimer. MRN has two DNA-binding modes, one ATP-dependent mode for loading onto DNA ends and one ATP-independent mode through Mre11's C terminus, suggesting how it may interact with DSBs and intact DNA. MRNs two 60-nm-long coiled-coil domains form a linear rod structure, the apex of which is assembled by the two joined zinc-hook motifs. Apices from two MRN complexes can further dimerize, forming 120-nm spanning MRN-MRN structures. Our results illustrate the architecture of MRN and suggest how it mechanistically integrates catalytic and tethering functions.

The Cause and Consequence of Replication Stress in Adult T-Cell Leukemia

EP300 and its homolog CBP are transcriptional co-activators of genes involved in a broad range of cellular processes, including proliferation, differentiation, apoptosis and DNA repair. Inactivating mutations in both EP300 and CBP have been reported in developmental disorders, such as Rubinstein-Taybi Syndrome, and several hematological malignancies. Most recently, somatic hypermutations in EP300 (but not CBP) were identified in Adult T-cell leukemia/lymphoma (ATLL) patients, diagnosed in Northern America. The mechanistic contribution of EP300 dysregulation to NA-ATLL etiology is currently unknown. ATLL is caused by infection by the Human T-cell Lymphotrophic Virus Type-1 (HTLV-1), that is endemic to Japan, the Caribbean basin, South America, southern parts of North America, Eastern Europe and certain areas of Africa. Unlike patients diagnosed in Japan (J-ATLL), NA-ATLL patient display a severe case of chemo-refractory disease with very poor prognosis. While prominent differences in the transcriptional and epigenetic landscape, particularly the p300 mutational status, has been identified between J-ATLL and NA-ATLL patients, the mechanistic contribution of p300 dysregulation to disease severity is yet to be established. Studies have revealed that the independent inhibition of EP300 in human cells results in a unique transcriptional landscape, involving the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Here, we show that EP300-mutated ATLL cells display a protracted S-phase, extensive genomic instability, dysregulations in DNA replication and repair dynamics. Using a powerful locus specific single molecule assay and genome-wide DNA fiber analysis, we show that perturbed DNA replication in EP300-mutated ATLL cells is characterized by elevated origin firing, increased replisome pausing and prominent nucleolytic degradation of nascent DNA at collapsed forks. The elevated origin firing in the absence of EP300 is likely driven by the inherent endogenous increase in c-MYC expression, which has been previously seen in EP300-mutated MCF7 cells. The striking replication pausing, fork collapse and nascent strand degradation observed in EP300-deficient NA-ATLL cells results in the persistence of single stranded DNA (ssDNA) genome-wide, resulting in the hyperphosphorylation of RPA at Ser4/8. Inhibition of Mre11 nuclease partially rescued the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Elevated endogenous genomic instability, observed in ATLL cells during the S-phase is exacerbated by EP300 deficiency, even in the absence of exogenous stress/damage. Analysis of the consequences of S-phase abnormalities and genomic instability revealed elevated micronuclei and cytosolic DNA in ATLL cells exposed to DNA damage. Preliminary studies evaluating the innate immune system activation (cGAS-STING-pIRF3) by immunoblotting, in response to stress, has led to some intriguing results. There is a clear separation in the ability of J-ATLL versus NA-ATLL cells in activating the innate immune system, a response that appears to be EP300 independent. These results raise the possibility of the contribution of a suppressed innate immune response, a phenomenon seen in many fast-progressing cancers, to the etiology of NA-ATLL. In addition to increasing our understanding of the mechanisms contributing to ATLL in general, the results from this study have helped identify new mechanism-based treatment regimens that could target the replicative and innate immune system vulnerabilities observed in North American ATLL patients.