Abstract
The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3'-5' exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3' single-strand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time- and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Acid Anhydride Hydrolases
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Alleles
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Amino Acid Sequence
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Ataxia Telangiectasia Mutated Proteins
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Base Sequence
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cell Line
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Cell Line, Tumor
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Colonic Neoplasms / genetics*
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Colonic Neoplasms / metabolism*
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DNA Mismatch Repair*
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DNA Repair Enzymes / genetics
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DNA Repair Enzymes / metabolism
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DNA Replication / genetics*
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DNA, Neoplasm / genetics
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DNA, Neoplasm / metabolism
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / genetics*
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DNA-Binding Proteins / metabolism*
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Genes, Dominant
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Humans
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MRE11 Homologue Protein
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Multiprotein Complexes
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Mutation*
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Phosphorylation
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Recombination, Genetic
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Thymidine / pharmacology
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Transfection
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Tumor Suppressor Proteins / genetics
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Tumor Suppressor Proteins / metabolism
Substances
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Cell Cycle Proteins
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DNA, Neoplasm
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DNA-Binding Proteins
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MRE11 protein, human
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Multiprotein Complexes
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NBN protein, human
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Nuclear Proteins
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Recombinant Proteins
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Tumor Suppressor Proteins
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ATM protein, human
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases
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MRE11 Homologue Protein
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Acid Anhydride Hydrolases
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RAD50 protein, human
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DNA Repair Enzymes
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Thymidine