A novel role for the peptidyl-prolyl cis-trans isomerase Cyclophilin A in DNA-repair following replication fork stalling via the MRE11-RAD50-NBS1 complex.

Cyclosporin A (CsA) induces DNA double-strand breaks in LIG4 syndrome fibroblasts, specifically upon transit through S-phase. The basis underlying this has not been described. CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair. CYPA is a peptidyl-prolyl cis-trans isomerase (PPI). CsA inhibits the PPI activity of CYPA. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression interaction analysis, we describe novel impacts of CYPA loss and inhibition on DNA repair. We characterise a direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 complex, providing evidence that CYPA influences DNA repair at the level of DNA end resection. We define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability. We explore examples of how CYPA inhibition may be exploited to selectively kill cancers sharing characteristic genomic instability profiles, including MYCN-driven Neuroblastoma, Multiple Myeloma and Chronic Myelogenous Leukaemia. These findings propose a repurposing strategy for Cyclophilin inhibitors.

Germline pathogenic variants in the MRE11, RAD50, and NBN (MRN) genes in cancer predisposition: A systematic review and meta-analysis.

The MRE11, RAD50, and NBN genes encode the MRN complex sensing DNA breaks and directing their repair. While carriers of biallelic germline pathogenic variants (gPV) develop rare chromosomal instability syndromes, the cancer risk in heterozygotes remains controversial. We performed a systematic review and meta-analysis of 53 studies in patients with different cancer diagnoses to better understand the cancer risk. We found an increased risk (odds ratio, 95% confidence interval) for gPV carriers in NBN for melanoma (7.14; 3.30-15.43), pancreatic cancer (4.03; 2.14-7.58), hematological tumors (3.42; 1.14-10.22), and prostate cancer (2.44, 1.84-3.24), but a low risk for breast cancer (1.29; 1.00-1.66) and an insignificant risk for ovarian cancer (1.53; 0.76-3.09). We found no increased breast cancer risk in carriers of gPV in RAD50 (0.93; 0.74-1.16; except of c.687del carriers) and MRE11 (0.87; 0.66-1.13). The secondary burden analysis compared the frequencies of gPV in MRN genes in patients from 150 studies with those in the gnomAD database. In NBN gPV carriers, this analysis additionally showed a high risk for brain tumors (5.06; 2.39-9.52), a low risk for colorectal (1.64; 1.26-2.10) and hepatobiliary (2.16; 1.02-4.06) cancers, and no risk for endometrial, and gastric cancer. The secondary burden analysis showed also a moderate risk for ovarian cancer (3.00; 1.27-6.08) in MRE11 gPV carriers, and no risk for ovarian and hepatobiliary cancers in RAD50 gPV carriers. These findings provide a robust clinical evidence of cancer risks to guide personalized clinical management in heterozygous carriers of gPV in the MRE11, RAD50, and NBN genes.

[Research Progress in the Roles of MRE11-RAD50-NBS1 Complex and Human Diseases].

DNA is susceptible to various factors in vitro and in vivo and experience different forms of damage,among which double-strand break(DSB)is a deleterious form.To maintain the stability of genetic information,organisms have developed multiple mechanisms to repair DNA damage.Among these mechanisms,homologous recombination(HR)is praised for the high accuracy.The MRE11-RAD50-NBS1(MRN)complex plays an important role in HR and is conserved across different species.The knowledge on the MRN complex mainly came from the previous studies in Saccharomyces cerevisiae and Caenorhabditis elegans,while studies in the last decades have revealed the role of mammalian MRN complex in DNA repair of higher animals.In this review,we first introduces the MRN complex regarding the composition,structure,and roles in HR.In addition,we discuss the human diseases such as ataxia-telangiectasia-like disorder,Nijmegen breakage syndrome,and Nijmegen breakage syndrome-like disorder that are caused by dysfunctions in the MRN complex.Furthermore,we summarize the mouse models established to study the clinical phenotypes of the above diseases.

A novel SIK2 inhibitor SIC-19 exhibits synthetic lethality with PARP inhibitors in ovarian cancer.

PURPOSE: Ovarian cancer patients with HR proficiency (HRP) have had limited benefits from PARP inhibitor treatment, highlighting the need for improved therapeutic strategies. In this study, we developed a novel SIK2 inhibitor, SIC-19, and investigated its potential to enhance the sensitivity and expand the clinical utility of PARP inhibitors in ovarian cancer. METHODS: The SIK2 protein was modeled using a Molecular Operating Environment (MOE), and the most favorable model was selected based on a GBVI/WSA dG scoring function. The Chembridge Compound Library was screened, and the top 20 candidate compounds were tested for their interaction with SIK2 and downstream substrates, AKT-pS473 and MYLK-pS343. SIC-19 emerged as the most promising drug candidate and was further evaluated using multiple assays. RESULTS: SIC-19 exhibited selective and potent inhibition of SIK2, leading to its degradation through the ubiquitination pathway. The IC50 of SIC-19 correlated inversely with endogenous SIK2 expression in ovarian cancer cell lines. Treatment with SIC-19 significantly inhibited cancer cell growth and sensitized cells to PARP inhibitors in vitro, as well as in ovarian cancer organoids and xenograft models. Mechanistically, SIK2 knockdown and SIC-19 treatment reduced RAD50 phosphorylation at Ser635, prevented nuclear translocation of RAD50, disrupted nuclear filament assembly, and impaired DNA homologous recombination repair, ultimately inducing apoptosis. These findings highlight the crucial role of SIK2 in the DNA HR repair pathway and demonstrate the significant PARP inhibitor sensitization achieved by SIC-19 in ovarian cancer. CONCLUSIONS: SIC-19, a novel SIK2 inhibitor, effectively inhibits tumor cell growth in ovarian cancer by interfering with RAD50-mediated DNA HR repair. Furthermore, SIC-19 enhances the efficacy of PARP inhibitors, providing a promising therapeutic strategy to improve outcomes for ovarian cancer patients.

The Association between Inefficient Repair of DNA Double-Strand Breaks and Common Polymorphisms of the HRR and NHEJ Repair Genes in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation affecting up to 2.0% of adults around the world. The molecular background of RA has not yet been fully elucidated, but RA is classified as a disease in which the genetic background is one of the most significant risk factors. One hallmark of RA is impaired DNA repair observed in patient-derived peripheral blood mononuclear cells (PBMCs). The aim of this study was to correlate the phenotype defined as the efficiency of DNA double-strand break (DSB) repair with the genotype limited to a single-nucleotide polymorphism (SNP) of DSB repair genes. We also analyzed the expression level of key DSB repair genes. The study population contained 45 RA patients and 45 healthy controls. We used a comet assay to study DSB repair after in vitro exposure to bleomycin in PBMCs from patients with rheumatoid arthritis. TaqMan SNP Genotyping Assays were used to determine the distribution of SNPs and the Taq Man gene expression assay was used to assess the RNA expression of DSB repair-related genes. PBMCs from patients with RA had significantly lower bleomycin-induced DNA lesion repair efficiency and we identified more subjects with inefficient DNA repair in RA compared with the control (84.5% vs. 24.4%; OR 41.4, 95% CI, 4.8-355.01). Furthermore, SNPs located within the RAD50 gene (rs1801321 and rs1801320) increased the OR to 53.5 (95% CI, 4.7-613.21) while rs963917 and rs3784099 (RAD51B) to 73.4 (95% CI, 5.3-1011.05). These results were confirmed by decision tree (DT) analysis (accuracy 0.84; precision 0.87, and specificity 0.86). We also found elevated expression of RAD51B, BRCA1, and BRCA2 in PBMCs isolated from RA patients. The findings indicated that impaired DSB repair in RA may be related to genetic variations in DSB repair genes as well as their expression levels. However, the mechanism of this relation, and whether it is direct or indirect, needs to be elucidated.

A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

Structural basis for plasmid restriction by SMC JET nuclease.

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.

Effects of RAD50 SNP, sodium intake, and H. pylori infection on gastric cancer survival in Korea.

BACKGROUND: Double-strand break repair protein (RAD50) gene plays important roles in genomic integrity, DNA double-strand break repair, cell cycle checkpoint activation, telomere maintenance, and meiotic recombination. The risk allele of RAD50 may negatively affect cancer by reducing the DNA repair capacity. Additionally, Sodium intake and Helicobacter pylori (H. pylori) infection are major risk factors for gastric cancer (GC). Our study investigated the association between polymorphisms in RAD50 gene and the risk of GC case-fatality. We evaluated whether the association differed with sodium intake or H. pylori infection. METHODS: We enrolled 490 patients from two hospitals between 2002 and 2006. Their survival or death was prospectively followed up until December 31, 2016, through a review of medical records and telephone surveys. The GC survival was assessed using the Cox proportional hazards regression analysis. RESULTS: In 319 GC cases, the total person-years were 1928.3, and the median survival years was 5.4 years. A total of 137 GC deaths were recorded. Our fully adjusted model showed that the GG type of RAD50 rs17772583 polymorphism is significantly associated with an increased risk of GC case-fatality (hazard ratio [HR] = 2.20, 95% confidence interval [CI] = 1.28-3.77) compared to that associated with the homozygous AA type. In the high sodium intake group, patients with the GG type of RAD50 rs17772583 showed a significantly higher GC case-fatality (HR = 8.61, 95% CI = 2.58-26.68) than that of patients with homozygous AA type. In the positive-H. pylori infection group, patients with GG-type RAD50 rs17772583 showed a significantly higher GC case-fatality (HR = 10.11, 95% CI = 2.81-36.35) than that of with AA homozygotes. CONCLUSIONS: Patients with GG-type RAD50 rs17772583, high sodium intake, or a positive-H. pylori infection are at a significantly increased risk of GC case-fatality compared to that associated with the absence of these risk factors.

Bone Marrow Failure and Immunodeficiency Associated with Human RAD50 Variants.

PURPOSE: The MRE11-RAD50-NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks. Pathogenic variants in NBN and MRE11 give rise to the autosomal-recessive diseases, Nijmegen breakage syndrome (NBS) and ataxia telangiectasia-like disorder, respectively. The clinical consequences of pathogenic variants in RAD50 are incompletely understood. We aimed to characterize a newly identified RAD50 deficiency/NBS-like disorder (NBSLD) patient with bone marrow failure and immunodeficiency. METHODS: We report on a girl with microcephaly, mental retardation, bird-like face, short stature, bone marrow failure and B-cell immunodeficiency. We searched for candidate gene by whole-exome sequencing and analyzed the cellular phenotype of patient-derived fibroblasts using immunoblotting, radiation sensitivity assays and lentiviral complementation experiments. RESULTS: Compound heterozygosity for two variants in the RAD50 gene (p.Arg83His and p.Glu485Ter) was identified in this patient. The expression of RAD50 protein and MRN complex formation was maintained in the cells derived from this patient. DNA damage-induced activation of the ATM kinase was markedly decreased, which was restored by the expression of wild-type (WT) RAD50. Radiosensitivity appeared inconspicuous in the patient-derived cell line as assessed by colony formation assay. The RAD50R83H missense substitution did not rescue the mitotic defect in complementation experiments using RAD50-deficient fibroblasts, whereas RAD50WT did. The RAD50E485X nonsense variant was associated with in-frame skipping of exon 10 (p.Glu485_545del). CONCLUSION: These findings indicate important roles of RAD50 in human bone marrow and immune cells. RAD50 deficiency/NBSLD can manifest as a distinct inborn error of immunity characterized by bone marrow failure and B-cell immunodeficiency.

Silencing TRIM29 Sensitizes Non-small Cell Lung Cancer Cells to Anlotinib by Promoting Apoptosis via Binding RAD50.

BACKGROUND: Previous studies have proposed that the transcriptional regulatory factor tripartite motif containing 29 (TRIM29) is involved in carcinogenesis via binding with nucleic acid. TRIM29 is confirmed to be highly expressed when the cancer cells acquire therapy-resistant properties. We noticed that TRIM29 levels were significantly increased in anlotinib-resistant NCI-H1975 (NCI-H1975/AR) cells via mining data information from gene expression omnibus (GEO) gene microarray (GSE142031; log2 fold change > 1, p < 0.05). OBJECTIVE: Our study aimed to investigate the function of TRIM29 on the resistance to anlotinib in non-small cell lung cancer (NSCLC) cells, including NCI-H1975 and A549 cells. METHODS: Real-time RT-PCR and western blot were used to detect TRIM29 expression in anlotinib-resistant NSCLC (NSCLC/AR) cells. Apoptosis were determined through flow cytometry, acridine orange/ethidium bromide staining as well as western blot. ELISA was used to measure the content of C-X3-C motif chemokine ligand 1. Co-Immunoprecipitation assay was performed to verify the interaction between TRIM29 and RAD50 double-strand break repair protein (RAD50). RESULTS: TRIM29 expression was shown to be elevated in the cytoplasm and nucleus of NSCLC/AR cells compared to normal NSCLC cells. Next, we demonstrated that TRIM29 knockdown facilitated apoptosis and enhanced the sensitivity to anlotinib in NSCLC/AR cells. Based on the refined results citing from the database BioGRID, it was proved that TRIM29 interacted with RAD50. Herein, RAD50 overexpression diminished the pro-apoptotic effect induced by silencing TRIM29 in anlotinib-resistant A549 (A549/AR) cells. CONCLUSION: Finally, we concluded that the increased sensitivity to anlotinib in NSCLC/AR cells was achieved by knocking down TRIM29, besides, the positive effects of TRIM29 knockdown were attributed to the promotion of apoptosis via binding to RAD50 in NSCLC/AR cell nucleus. Therefore, TRIM29 might become a potential target for overcoming anlotinib resistance in NSCLC treatment.

SMC-based immunity against extrachromosomal DNA elements.

SMC and SMC-like complexes promote chromosome folding and genome maintenance in all domains of life. Recently, they were also recognized as factors in cellular immunity against foreign DNA. In bacteria and archaea, Wadjet and Lamassu are anti-plasmid/phage defence systems, while Smc5/6 and Rad50 complexes play a role in anti-viral immunity in humans. This raises an intriguing paradox - how can the same, or closely related, complexes on one hand secure the integrity and maintenance of chromosomal DNA, while on the other recognize and restrict extrachromosomal DNA? In this minireview, we will briefly describe the latest understanding of each of these complexes in immunity including speculations on how principles of SMC(-like) function may explain how the systems recognize linear or circular forms of invading DNA.

Drug repurposing of propafenone to discover novel anti-tumor agents by impairing homologous recombination to delay DNA damage recovery of rare disease conjunctival melanoma.

Conjunctival melanoma (CM), a rare and fatal malignant ocular tumor, lacks proper diagnostic biomarkers and therapy. Herein, we revealed the novel application of propafenone, an FDA-approved antiarrhythmic medication, which was identified effective in inhibiting CM cells viability and homologous recombination pathway. Detailed structure-activity relationships generated D34 as one of the most promising derivatives, which strongly suppressed the proliferation, viability, and migration of CM cells at submicromolar concentrations. Mechanically, D34 had the potential to increase γ-H2AX nuclear foci and aggravated DNA damage by suppressing homologous recombination pathway and its factors, particularly the complex of MRE11-RAD50-NBS1. D34 bound to human recombinant MRE11 protein and inhibited its endonuclease activity. Moreover, D34 dihydrochloride significantly suppressed tumor growth in the CRMM1 NCG xenograft model without obvious toxicity. Our finding shows that propafenone derivatives modulating the MRE11-RAD50-NBS1 complex will most likely provide an approach for CM targeted therapy, especially for improving chemo- and radio-sensitivity for CM patients.

Importance of Germline and Somatic Alterations in Human MRE11, RAD50, and NBN Genes Coding for MRN Complex.

The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.

Mre11-Rad50: the DNA end game.

The Mre11-Rad50-(Nbs1/Xrs2) complex is an evolutionarily conserved factor for the repair of DNA double-strand breaks and other DNA termini in all kingdoms of life. It is an intricate DNA associated molecular machine that cuts, among other functions, a large variety of free and obstructed DNA termini for DNA repair by end joining or homologous recombination, yet leaves undamaged DNA intact. Recent years have brought progress in both the structural and functional analyses of Mre11-Rad50 orthologs, revealing mechanisms of DNA end recognition, endo/exonuclease activities, nuclease regulation and DNA scaffolding. Here, I review our current understanding and recent progress on the functional architecture Mre11-Rad50 and how this chromosome associated coiled-coil ABC ATPase acts as DNA topology specific endo-/exonuclease.

Deubiquitinase USP2 stabilizes the MRE11-RAD50-NBS1 complex at DNA double-strand break sites by counteracting the ubiquitination of NBS1.

The MRE11-RAD50-NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.

FOXO3a Mediates Homologous Recombination Repair (HRR) via Transcriptional Activation of MRE11, BRCA1, BRIP1, and RAD50.

To test whether homologous recombination repair (HRR) depends on FOXO3a, a cellular aging model of human dermal fibroblast (HDF) and tet-on flag-h-FOXO3a transgenic mice were studied. HDF cells transfected with over-expression of wt-h-FOXO3a increased the protein levels of MRE11, BRCA1, BRIP1, and RAD50, while knock-down with siFOXO3a decreased them. The protein levels of MRE11, BRCA1, BRIP1, RAD50, and RAD51 decreased during cellular aging. Chromatin immunoprecipitation (ChIP) assay was performed on FOXO3a binding accessibility to FOXO consensus sites in human MRE11, BRCA1, BRIP1, and RAD50 promoters; the results showed FOXO3a binding decreased during cellular aging. When the tet-on flag-h-FOXO3a mice were administered doxycycline orally, the protein and mRNA levels of flag-h-FOXO3a, MRE11, BRCA1, BRIP1, and RAD50 increased in a doxycycline-dose-dependent manner. In vitro HRR assays were performed by transfection with an HR vector and I-SceI vector. The mRNA levels of the recombined GFP increased after doxycycline treatment in MEF but not in wt-MEF, and increased in young HDF comparing to old HDF, indicating that FOXO3a activates HRR. Overall, these results demonstrate that MRE11, BRCA1, BRIP1, and RAD50 are transcriptional target genes for FOXO3a, and HRR activity is increased via transcriptional activation of MRE11, BRCA1, BRIP1, and RAD50 by FOXO3a.

Somatic NGS Analysis of DNA Damage Response (DDR) Genes ATM, MRE11A, RAD50, NBN, and ATR in Locally Advanced Rectal Cancer Treated with Neoadjuvant Chemo-Radiotherapy.

BACKGROUND: Neoadjuvant chemo-radiotherapy (nCRT) represents the standard of care for locally advanced rectal cancer (LARC); however, there exists no biomarker that can predict the cancer's response to treatment as less than 20% of patients experience pathological complete response (pCR). Ionizing radiations induce double strand breaks (DSBs) and trigger a DNA damage response (DDR) involving ATM, ATR, and the MRN complex (MRE11, Rad50, and NBS1). In this study, we performed an extensive mutational analysis of the genes involved in the DDR pathway in LARC patients who have undergone nCRT. METHODS: 13 LARC patients with pCR and 11 LARC patients with partial response (pPR) were investigated using a NGS dedicated panel, designed for formalin-fixed paraffin-embedded (FFPE) samples, containing ATR, ATM, and MRE11-RAD50-NBN genes. The identified variants were classified according to guidelines' recommendations. RESULTS: Eight non-benign variants, six of which were observed in 3 (23%) out of 13 pCR patients, were identified. In particular, a pCR patient carried out a pathogenetic frameshift mutation in exon 21 of the RAD50 gene. The two remaining non-benign missense variants were found in 2 (18%) out of 11 patients in the pPR group. CONCLUSIONS: Our data show that the genes involved in the Homologous Recombination (HR) pathway are rarely mutated in LARC; however, given the identification of a missense mutation in RAD 50 in one case of pCR, it could be worth exploring its potential role as a biomarker in larger series.

Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation.

The metal binding at protein-protein interfaces is still uncharted territory in intermolecular interactions. To date, only a few protein complexes binding Zn(II) in an intermolecular manner have been deeply investigated. The most notable example of such interfaces is located in the highly conserved Rad50 protein, part of the Mre11-Rad50-Nbs1 (MRN) complex, where Zn(II) is required for homodimerization (Zn(Rad50)2). The high stability of Zn(Rad50)2 is conserved not only for the protein derived from the thermophilic archaeon Pyrococcus furiosus (logK12 = 20.95 for 130-amino-acid-long fragment), which was the first one studied, but also for the human paralog studied here (logK12 = 19.52 for a 183-amino-acid-long fragment). As we reported previously, the extremely high stability results from the metal-coupled folding process where particular Rad50 protein fragments play a critical role. The sequence-structure-stability analysis based on human Rad50 presented here separates the individual structural components that increase the stability of the complex, pointing to amino acid residues far away from the Zn(II) binding site as being largely responsible for the complex stabilization. The influence of the individual components is very well reflected by the previously published crystal structure of the human Rad50 zinc hook (PDB: 5GOX). In addition, we hereby report the effect of phosphorylation of the zinc hook domain, which exerts a destabilizing effect on the domain. This study identifies factors governing the stability of metal-mediated protein-protein interactions and illuminates their molecular basis.

Requirements for MRN endonuclease processing of topoisomerase II-mediated DNA damage in mammalian cells.

During a normal topoisomerase II (TOP2) reaction, the enzyme forms a covalent enzyme DNA intermediate consisting of a 5' phosphotyrosyl linkage between the enzyme and DNA. While the enzyme typically rejoins the transient breakage after strand passage, a variety of conditions including drugs targeting TOP2 can inhibit DNA resealing, leading to enzyme-mediated DNA damage. A critical aspect of the repair of TOP2-mediated damage is the removal of the TOP2 protein covalently bound to DNA. While proteolysis plays a role in repairing this damage, nucleolytic enzymes must remove the phosphotyrosyl-linked peptide bound to DNA. The MRN complex has been shown to participate in the removal of TOP2 protein from DNA following cellular treatment with TOP2 poisons. In this report we used an optimized ICE (In vivo Complex of Enzyme) assay to measure covalent TOP2/DNA complexes. In agreement with previous independent reports, we find that the absence or inhibition of the MRE11 endonuclease results in elevated levels of both TOP2α and TOP2ß covalent complexes. We also examined levels of TOP2 covalent complexes in cells treated with the proteasome inhibitor MG132. Although MRE11 inhibition plus MG132 was not synergistic in etoposide-treated cells, ectopic overexpression of MRE11 resulted in removal of TOP2 even in the presence of MG132. We also found that VCP/p97 inhibition led to elevated TOP2 covalent complexes and prevented the removal of TOP2 covalent complexes by MRE11 overexpression. Our results demonstrate the existence of multiple pathways for proteolytic processing of TOP2 prior to nucleolytic processing, and that MRE11 can process TOP2 covalent complexes even when the proteasome is inhibited. The interactions between VCP/p97 and proteolytic processing of TOP2 covalent complexes merit additional investigation.

Structural mechanism of endonucleolytic processing of blocked DNA ends and hairpins by Mre11-Rad50.

DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood but physiologically critical endonuclease activity by the Mre11-Rad50 complex. Here, we report cryoelectron microscopy (cryo-EM) structures of the bacterial Mre11-Rad50 homolog SbcCD bound to a protein-blocked DNA end and a DNA hairpin. The structures reveal that Mre11-Rad50 bends internal DNA for endonucleolytic cleavage and show how internal DNA, DNA ends, and hairpins are processed through a similar ATP-regulated conformational state. Furthermore, Mre11-Rad50 is loaded onto blocked DNA ends with Mre11 pointing away from the block, explaining the distinct biochemistries of 3' → 5' exonucleolytic and endonucleolytic incision through the way Mre11-Rad50 interacts with diverse DNA ends. In summary, our results unify Mre11-Rad50's enigmatic nuclease diversity within a single structural framework and reveal how blocked DNA ends and hairpins are processed.