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1.
Cancer Res ; 77(19): 5327-5338, 2017 10 01.
Article in English | MEDLINE | ID: mdl-28819025

ABSTRACT

Hypomorphic mutations in the genes encoding the MRE11/RAD50/NBS1 (MRN) DNA repair complex lead to cancer-prone syndromes. MRN binds DNA double-strand breaks, where it functions in repair and triggers cell-cycle checkpoints via activation of the ataxia-telangiectasia mutated kinase. To gain understanding of MRN in cancer, we engineered mice with B lymphocytes lacking MRN, or harboring MRN in which MRE11 lacks nuclease activities. Both forms of MRN deficiency led to hallmarks of cancer, including oncogenic translocations involving c-Myc and the immunoglobulin locus. These preneoplastic B lymphocytes did not progress to detectable B lineage lymphoma, even in the absence of p53. Moreover, Mre11 deficiencies prevented tumorigenesis in a mouse model strongly predisposed to spontaneous B-cell lymphomas. Our findings indicate that MRN cannot be considered a standard tumor suppressor and instead imply that nuclease activities of MRE11 are required for oncogenesis. Inhibition of MRE11 nuclease activity increased DNA damage and selectively induced apoptosis in cells overexpressing oncogenes, suggesting MRE11 serves an important role in countering oncogene-induced replication stress. Thus, MRE11 may offer a target for cancer therapeutic development. More broadly, our work supports the idea that subtle enhancements of endogenous genome instability can exceed the tolerance of cancer cells and be exploited for therapeutic ends. Cancer Res; 77(19); 5327-38. ©2017 AACR.


Subject(s)
B-Lymphocytes/pathology , Cell Transformation, Neoplastic/pathology , DNA Repair Enzymes/physiology , DNA Replication , DNA-Binding Proteins/physiology , Lymphoma, B-Cell/pathology , Proto-Oncogene Proteins c-myc/metabolism , ATP-Binding Cassette Transporters/physiology , Acid Anhydride Hydrolases , Animals , Apoptosis , Ataxia Telangiectasia Mutated Proteins/metabolism , B-Lymphocytes/metabolism , Cell Cycle Proteins/physiology , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cells, Cultured , DNA Breaks, Double-Stranded , DNA Repair , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Fibroblasts/metabolism , Fibroblasts/pathology , Genomic Instability , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/metabolism , MRE11 Homologue Protein , Mice , Mutation , Nuclear Proteins/physiology , Oncogenes , Proto-Oncogene Proteins c-myc/genetics
2.
Nat Struct Mol Biol ; 22(9): 736-43, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26280532

ABSTRACT

Two kinases, ATM and DNA-PKcs, control rapid responses to DNA double-strand breaks (DSBs). The paradigm for ATM control is recruitment and activation by the Mre11-Rad50-NBS1 (MRN) sensor complex, whereas DNA-PKcs requires the sensor Ku (Ku70-Ku80). Using mouse cells containing targeted mutant alleles of Mre11 (Mre11a) and/or Ku70 (Xrcc6), together with pharmacologic kinase inhibition, we demonstrate that ATM can be activated by DSBs in the absence of MRN. When MRN is deficient, DNA-PKcs efficiently substitutes for ATM in facilitating local chromatin responses. In the absence of both MRN and Ku, ATM is recruited to chromatin, where it phosphorylates H2AX and triggers the G2-M cell-cycle checkpoint, but the DNA-repair functions of MRN are not restored. These results suggest that, in contrast to straightforward recruitment and activation by MRN, a complex interplay between sensors has a substantial role in ATM control.


Subject(s)
DNA Breaks, Double-Stranded , DNA/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Mice , Protein Binding
3.
Hum Mol Genet ; 22(25): 5146-59, 2013 Dec 20.
Article in English | MEDLINE | ID: mdl-23912341

ABSTRACT

DNA double-strand breaks (DSBs) can lead to instability of the genome if not repaired correctly. The MRE11/RAD50/NBS1 (MRN) complex binds DSBs and initiates damage-induced signaling cascades via activation of the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia- and rad3-related (ATR) kinases. Mutations throughout MRE11 cause ataxia-telangiectasia-like disorder (ATLD) featuring cerebellar degeneration, and cancer-predisposition in certain kindreds. Here, we have examined the impact on DNA damage signaling of several disease-associated MRE11A alleles to gain greater understanding of the mechanisms underlying the diverse disease sequelae of ATLD. To this end, we have designed a system whereby endogenous wild-type Mre11a is conditionally deleted and disease-associated MRE11 mutants are stably expressed at physiologic levels. We find that mutations in the highly conserved N-terminal domain impact ATM signaling by perturbing both MRE11 interaction with NBS1 and MRE11 homodimerization. In contrast, an inherited allele in the MRE11 C-terminus maintains MRN interactions and ATM/ATR kinase activation. These findings reveal that ATLD patients have reduced ATM activation resulting from at least two distinct mechanisms: (i) N-terminal mutations destabilize MRN interactions, and (ii) mutation of the extreme C-terminus maintains interactions but leads to low levels of the complex. The N-terminal mutations were found in ATLD patients with childhood cancer; thus, our studies suggest a clinically relevant dichotomy in MRE11A alleles. More broadly, these studies underscore the importance of understanding specific effects of hypomorphic disease-associated mutations to achieve accurate prognosis and appropriate long-term medical surveillance.


Subject(s)
Ataxia Telangiectasia/genetics , DNA-Binding Proteins/genetics , Neoplasms/genetics , Spinocerebellar Degenerations/genetics , Alleles , Ataxia Telangiectasia/etiology , Ataxia Telangiectasia/physiopathology , DNA Breaks, Double-Stranded , DNA Damage/genetics , Genetic Predisposition to Disease , Genomic Instability , Humans , MRE11 Homologue Protein , Mutation , Neoplasms/etiology , Neoplasms/pathology , Signal Transduction , Spinocerebellar Degenerations/physiopathology
4.
Nat Struct Mol Biol ; 19(2): 246-52, 2012 Jan 08.
Article in English | MEDLINE | ID: mdl-22231403

ABSTRACT

Homologous recombination facilitates accurate repair of DNA double-strand breaks (DSBs) during the S and G2 phases of the cell cycle by using intact sister chromatids as sequence templates. Homologous recombination capacity is maximized in S and G2 by cyclin-dependent kinase (CDK) phosphorylation of CtIP, which subsequently interacts with BRCA1 and the Mre11-Rad50-NBS1 (MRN) complex. Here we show that, in human and mouse, Mre11 controls these events through a direct interaction with CDK2 that is required for CtIP phosphorylation and BRCA1 interaction in normally dividing cells. CDK2 binds the C terminus of Mre11, which is absent in an inherited allele causing ataxia telangiectasia-like disorder. This newly uncovered role for Mre11 does not require ATM activation or nuclease activities. Therefore, functions of MRN are not restricted to DNA damage responses but include regulating homologous recombination capacity during the normal mammalian cell cycle.


Subject(s)
Carrier Proteins/metabolism , Cyclin-Dependent Kinase 2/metabolism , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Cell Cycle Proteins/metabolism , DNA Repair Enzymes/metabolism , Endodeoxyribonucleases , Humans , MRE11 Homologue Protein , Mice , Mice, Knockout , Phosphorylation , Protein Binding , Protein Interaction Mapping , Recombination, Genetic
5.
Nat Struct Mol Biol ; 16(8): 808-13, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19633670

ABSTRACT

The Mre11-Rad50-NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN complex in B lymphocytes or that possess an intact complex that harbors mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.


Subject(s)
B-Lymphocytes/metabolism , DNA Repair , Immunoglobulin Class Switching , Signal Transduction/physiology , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Acid Anhydride Hydrolases , Adaptor Proteins, Signal Transducing , Animals , Ataxia Telangiectasia Mutated Proteins , B-Lymphocytes/cytology , Base Sequence , Blotting, Western , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Proliferation , Cells, Cultured , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Flow Cytometry , Histones/genetics , Histones/metabolism , Immunoglobulin Heavy Chains/genetics , In Situ Hybridization, Fluorescence , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , MRE11 Homologue Protein , Male , Mice , Mice, Knockout , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Recombination, Genetic , Signal Transduction/genetics , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
6.
Cell ; 135(1): 85-96, 2008 Oct 03.
Article in English | MEDLINE | ID: mdl-18854157

ABSTRACT

The Mre11/Rad50/NBS1 (MRN) complex maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution but play unknown roles in mammals. To define the functions of Mre11, we engineered targeted mouse alleles that either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from the absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology-directed double-strand-break repair and a contributing role in activating the ATR kinase. However, the nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair, distinct from MRN control of ATM signaling.


Subject(s)
DNA Repair Enzymes/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Genomic Instability , Amino Acid Sequence , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Cell Line, Transformed , Cell Proliferation , DNA Breaks, Double-Stranded , DNA Damage , DNA Repair Enzymes/chemistry , DNA-Binding Proteins/chemistry , Fibroblasts/metabolism , MRE11 Homologue Protein , Mice , Protein Serine-Threonine Kinases/metabolism , Recombination, Genetic , Telomere/metabolism , Tumor Suppressor Proteins/metabolism
7.
J Biol Chem ; 281(36): 25994-6003, 2006 Sep 08.
Article in English | MEDLINE | ID: mdl-16829680

ABSTRACT

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , DNA Repair , Proteins/metabolism , S-Adenosylmethionine/metabolism , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , DNA/chemistry , DNA/metabolism , Deoxyadenosines/metabolism , Deoxyribodipyrimidine Photo-Lyase/genetics , Deoxyribodipyrimidine Photo-Lyase/metabolism , Iron/metabolism , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/isolation & purification , Iron-Sulfur Proteins/metabolism , Molecular Structure , Proteins/genetics , Proteins/isolation & purification , S-Adenosylmethionine/chemistry , Sulfides/metabolism , Thymine/analogs & derivatives , Thymine/metabolism
8.
Arch Biochem Biophys ; 433(1): 288-96, 2005 Jan 01.
Article in English | MEDLINE | ID: mdl-15581584

ABSTRACT

Pyruvate formate lyase activating enzyme is a member of a novel superfamily of enzymes that utilize S-adenosylmethionine to initiate radical catalysis. This enzyme has been isolated with several different iron-sulfur clusters, but single turnover monitored by EPR has identified the [4Fe-4S](1+) cluster as the catalytically active cluster; this cluster is believed to be oxidized to the [4Fe-4S](2+) state during turnover. The [4Fe-4S] cluster is coordinated by a three-cysteine motif common to the radical/S-adenosylmethionine superfamily, suggesting the presence of a unique iron in the cluster. The unique iron site has been confirmed by Mossbauer and ENDOR spectroscopy experiments, which also provided the first evidence for direct coordination of S-adenosylmethionine to an iron-sulfur cluster, in this case the unique iron of the [4Fe-4S] cluster. Coordination to the unique iron anchors the S-adenosylmethionine in the active site, and allows for a close association between the sulfonium of S-adenosylmethionine and the cluster as observed by ENDOR spectroscopy. The evidence to date leads to a mechanistic proposal involving inner-sphere electron transfer from the cluster to the sulfonium of S-adenosylmethionine, followed by or concomitant with C-S bond homolysis to produce a 5'-deoxyadenosyl radical; this transient radical abstracts a hydrogen atom from G734 to activate pyruvate formate lyase.


Subject(s)
Enzymes/chemistry , Enzymes/metabolism , Free Radicals/chemistry , Iron-Sulfur Proteins/chemistry , Acetyltransferases , Binding Sites , Catalysis , Electron Spin Resonance Spectroscopy , Electron Transport , Enzyme Activation , Iron-Sulfur Proteins/metabolism , S-Adenosylmethionine/metabolism , Spectroscopy, Mossbauer , Substrate Specificity
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