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1.
Chem Biol ; 20(1): 55-62, 2013 Jan 24.
Article in English | MEDLINE | ID: mdl-23352139

ABSTRACT

The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM function in cells, we undertook a high throughput screen of a chemical compound library for small molecule inhibitors of BLM. We present ML216, a potent inhibitor of the DNA unwinding activity of BLM. ML216 shows cell-based activity and can induce sister chromatid exchanges, enhance the toxicity of aphidicolin, and exert antiproliferative activity in cells expressing BLM, but not those lacking BLM. These data indicate that ML216 shows strong selectivity for BLM in cultured cells. We discuss the potential utility of such a BLM-targeting compound as an anticancer agent.


Subject(s)
Chromosomal Instability/drug effects , RecQ Helicases/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line , Cell Proliferation/drug effects , DNA/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays , Humans , Protein Binding/drug effects , RecQ Helicases/metabolism
2.
EMBO J ; 30(4): 692-705, 2011 Feb 16.
Article in English | MEDLINE | ID: mdl-21240188

ABSTRACT

Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJ-deficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ-BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an 'undamaged' duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress.


Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Bloom Syndrome/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Fanconi Anemia/genetics , RecQ Helicases/metabolism , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Cell Nucleus/metabolism , Cells, Cultured , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Helicases/physiology , DNA Replication/genetics , DNA Replication/physiology , Fanconi Anemia Complementation Group Proteins/genetics , Genomic Instability/genetics , HeLa Cells , Humans , Insecta , Protein Binding/physiology , Protein Interaction Mapping , RecQ Helicases/genetics , Tissue Distribution
3.
Science ; 329(5988): 219-23, 2010 Jul 09.
Article in English | MEDLINE | ID: mdl-20538911

ABSTRACT

A conserved DNA repair response is defective in the human genetic illness Fanconi anemia (FA). Mutation of some FA genes impairs homologous recombination and error-prone DNA repair, rendering FA cells sensitive to DNA cross-linking agents. We found a genetic interaction between the FA gene FANCC and the nonhomologous end joining (NHEJ) factor Ku70. Disruption of both FANCC and Ku70 suppresses sensitivity to cross-linking agents, diminishes chromosome breaks, and reverses defective homologous recombination. Ku70 binds directly to free DNA ends, committing them to NHEJ repair. We show that purified FANCD2, a downstream effector of the FA pathway, might antagonize Ku70 activity by modifying such DNA substrates. These results reveal a function for the FA pathway in processing DNA ends, thereby diverting double-strand break repair away from abortive NHEJ and toward homologous recombination.


Subject(s)
Antigens, Nuclear/genetics , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/genetics , Fanconi Anemia Complementation Group C Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , Recombination, Genetic , Animals , Antigens, Nuclear/metabolism , Cell Line , Chickens , Chromosome Breakage , Cross-Linking Reagents/pharmacology , DNA-Binding Proteins/metabolism , Fanconi Anemia Complementation Group C Protein/metabolism , Fanconi Anemia Complementation Group D2 Protein/chemistry , Fanconi Anemia Complementation Group D2 Protein/genetics , Gene Conversion , Genes, Immunoglobulin , Humans , Immunoglobulin M/genetics , Ku Autoantigen , Point Mutation , Recombinant Proteins/metabolism
4.
J Biol Chem ; 283(52): 36132-9, 2008 Dec 26.
Article in English | MEDLINE | ID: mdl-18978354

ABSTRACT

Fanconi anemia (FA) is a heritable human cancer-susceptibility disorder, delineating a genetically heterogenous pathway for the repair of replication-blocking lesions such as interstrand DNA cross-links. Here we demonstrate that one component of this pathway, FANCJ, is a structure-specific DNA helicase that dissociates guanine quadruplex DNA (G4 DNA) in vitro. Moreover, in contrast with previously identified G4 DNA helicases, such as the Bloom's helicase (BLM), FANCJ unwinds G4 substrates with 5'-3' polarity. In the FA-J human patient cell line EUFA0030 the loss of FANCJ G4 unwinding function correlates with the accumulation of large genomic deletions in the vicinity of sequences, which match the G4 DNA signature. Together these findings support a role for FANCJ in the maintenance of potentially unstable genomic G/C tracts during replication.


Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , DNA Helicases/metabolism , Fanconi Anemia Complementation Group Proteins/metabolism , G-Quadruplexes , RecQ Helicases/metabolism , Binding, Competitive , Cell Line , Cell Line, Tumor , Cross-Linking Reagents/pharmacology , DNA Replication , Gene Deletion , Genetic Predisposition to Disease , Genome , Humans , Nucleic Acid Conformation , Nucleic Acid Hybridization
5.
J Biol Chem ; 283(26): 17766-76, 2008 Jun 27.
Article in English | MEDLINE | ID: mdl-18448429

ABSTRACT

RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3'-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1-56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells.


Subject(s)
DNA Helicases/chemistry , DNA/chemistry , G-Quadruplexes , RecQ Helicases/chemistry , RecQ Helicases/physiology , Adenosine Triphosphatases/chemistry , Base Sequence , Humans , Molecular Sequence Data , Mutagenesis , Protein Binding , Protein Conformation , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Substrate Specificity
6.
Mol Cell Biol ; 27(24): 8421-30, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17938197

ABSTRACT

The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.


Subject(s)
Chromatin/enzymology , Fanconi Anemia Complementation Group D2 Protein/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination , Animals , Catalytic Domain , Cell Cycle , Cell Line , Chickens , DNA Damage , Fanconi Anemia Complementation Group L Protein/metabolism , Humans , Protein Binding
7.
Nat Struct Mol Biol ; 12(9): 763-71, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16116434

ABSTRACT

The helicase-associated endonuclease for fork-structured DNA (Hef) is an archaeabacterial protein that processes blocked replication forks. Here we have isolated the vertebrate Hef ortholog and investigated its molecular function. Disruption of this gene in chicken DT40 cells results in genomic instability and sensitivity to DNA cross-links. The similarity of this phenotype to that of cells lacking the Fanconi anemia-related (FA) tumor-suppressor genes led us to investigate whether Hef functions in this pathway. Indeed, we found a genetic interaction between the FANCC and Hef genes. In addition, Hef is a component of the FA nuclear protein complex that facilitates its DNA damage-inducible chromatin localization and the monoubiquitination of the FA protein FANCD2. Notably, Hef interacts directly with DNA structures that are intermediates in DNA replication. This discovery sheds light on the origins, regulation and molecular function of the FA tumor-suppressor pathway in the maintenance of genome stability.


Subject(s)
Avian Proteins/metabolism , Cell Cycle Proteins/metabolism , Chickens , Conserved Sequence , DNA-Binding Proteins/metabolism , Fanconi Anemia/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Adenosine Triphosphatases/metabolism , Animals , Avian Proteins/chemistry , Avian Proteins/deficiency , Avian Proteins/genetics , Cell Cycle Proteins/genetics , Cell Line , Chickens/genetics , Chickens/metabolism , DNA/metabolism , DNA Damage , DNA Helicases/metabolism , DNA Repair , DNA Replication , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Endonucleases/genetics , Endonucleases/metabolism , Evolution, Molecular , Fanconi Anemia/genetics , Fanconi Anemia Complementation Group C Protein , Fanconi Anemia Complementation Group Proteins , Genomic Instability , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Tumor Suppressor Proteins/genetics
8.
Mol Cell ; 15(4): 607-20, 2004 Aug 27.
Article in English | MEDLINE | ID: mdl-15327776

ABSTRACT

The Fanconi anemia (FA) protein FANCC is essential for chromosome stability in vertebrate cells, a feature underscored by the extreme sensitivity of FANCC-deficient cells to agents that crosslink DNA. However, it is not known how this FA protein facilitates the repair of both endogenously acquired and mutagen-induced DNA damage. Here, we use the model vertebrate cell line DT40 to address this question. We discover that apart from functioning in homologous recombination, FANCC also promotes the mutational repair of endogenously generated abasic sites. Moreover in these vertebrate cells, the efficient repair of crosslinks requires the combined functions of FANCC, translesion synthesis, and homologous recombination. These studies reveal that the FA proteins cooperate with key mutagenesis and repair processes that enable replication of damaged DNA.


Subject(s)
Cell Cycle Proteins , DNA Repair , DNA-Binding Proteins , Proteins/genetics , Recombination, Genetic , Animals , Cell Line , Chickens , Chromosomal Instability , Cross-Linking Reagents/metabolism , Cross-Linking Reagents/pharmacology , DNA/drug effects , DNA/radiation effects , DNA Damage , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Epistasis, Genetic , Fanconi Anemia , Fanconi Anemia Complementation Group C Protein , Fanconi Anemia Complementation Group D2 Protein , Fanconi Anemia Complementation Group Proteins , Humans , Nuclear Proteins/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , X-Rays
9.
EMBO J ; 21(13): 3414-23, 2002 Jul 01.
Article in English | MEDLINE | ID: mdl-12093742

ABSTRACT

The Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identified FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.


Subject(s)
Cell Cycle Proteins , Fanconi Anemia/metabolism , Nuclear Proteins/physiology , Active Transport, Cell Nucleus , Amino Acid Substitution , Animals , Bacterial Proteins/analysis , COS Cells , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/ultrastructure , Chlorocebus aethiops , Chromosome Breakage , DNA-Binding Proteins/metabolism , Fanconi Anemia/genetics , Fanconi Anemia Complementation Group C Protein , Fanconi Anemia Complementation Group D2 Protein , Fanconi Anemia Complementation Group E Protein , Fanconi Anemia Complementation Group F Protein , Fanconi Anemia Complementation Group G Protein , Fanconi Anemia Complementation Group Proteins , HeLa Cells , Humans , Luminescent Proteins/analysis , Macromolecular Substances , Mutation, Missense , Nuclear Proteins/metabolism , Protein Binding , Protein Interaction Mapping , Proteins/genetics , Proteins/metabolism , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship
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