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Nucleic Acids Res. 2015 Mar 11;43(5):2678-90. doi: 10.1093/nar/gkv084. Epub 2015 Feb 11.

Replication fork integrity and intra-S phase checkpoint suppress gene amplification.

Author information

1
Department of Molecular Genetics, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
2
Department of Molecular Genetics, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA Department of Surgery, Cedars-Sinai Medical Center, West Hollywood, CA, USA.
3
Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
4
Department of Biology, San Diego State University, San Diego, CA 92182, USA.
5
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.

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.

PMID:
25672394
PMCID:
PMC4357702
DOI:
10.1093/nar/gkv084
[Indexed for MEDLINE]
Free PMC Article

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