title: Replication fork integrity and intra-S phase checkpoint suppress gene amplification.
abstract: Gene amplification is a phenotype-causing form of chromosome instability and is initiated by DNA double-strand breaks (DSBs). Cells with mutant p53 lose G1/S checkpoint and are permissive to gene amplification. In this study we show that mammalian cells become proficient for spontaneous gene amplification when the function of the DSB repair protein complex MRN (Mre11/Rad50/Nbs1) is impaired. Cells with impaired MRN complex experienced severe replication stress and gained substrates for gene amplification during replication, as evidenced by the increase of replication-associated single-stranded breaks that were converted to DSBs most likely through replication fork reversal. Impaired MRN complex directly compromised ATM/ATR-mediated checkpoints and allowed cells to progress through cell cycle in the presence of DSBs. Such compromised intra-S phase checkpoints promoted gene amplification independently from mutant p53. Finally, cells adapted to endogenous replication stress by globally suppressing genes for DNA replication and cell cycle progression. Our results indicate that the MRN complex suppresses gene amplification by stabilizing replication forks and by securing DNA damage response to replication-associated DSBs.
authors: Department of Molecular Genetics, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
authors: Anna
authors: A
authors: Kondratova
authors: Department of Molecular Genetics, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA Department of Surgery, Cedars-Sinai Medical Center, West Hollywood, CA, USA.
authors: Takaaki
authors: T
authors: Watanabe
authors: Department of Molecular Genetics, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
authors: Michael
authors: M
authors: Marotta
authors: Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
authors: Matthew
authors: M
authors: Cannon
authors: Department of Biology, San Diego State University, San Diego, CA 92182, USA.
authors: Anca M
authors: AM
authors: Segall
authors: Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
authors: David
authors: D
authors: Serre
authors: Department of Molecular Genetics, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA Department of Surgery, Cedars-Sinai Medical Center, West Hollywood, CA, USA Hisashi.Tanaka@cshs.org.
authors: Hisashi
authors: H
authors: Tanaka
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005784:Gene Amplification; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D059807:S Phase Cell Cycle Checkpoints / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D013762:Tetrahydrofolate Dehydrogenase; D064007:Ataxia Telangiectasia Mutated Proteins; D045643:DNA Repair Enzymes