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.
Highlights
Increase in the copy number of genomic segments harboring oncogenes and therapy target genes causes aggressive tumor phenotypes by promoting cancer progression and acquired therapy resistance [1,2,3,4]
DNA sequences for oligos used in this study are available upon request. pLSLP-CHOshMre11–562 was transfected into 293T cells with two other vectors to produce lentiviral particles that were infected into D229IRlox2–35-noIR-2 (D229IRlox2–35-Mre11KD) [9]
Fourteen days after lentiviral infection, we recovered a population of cells with significantly reduced expression of Mre11 protein (Mre11-KD cells), relatively to the control cells expressing either non-targeting shRNA or green fluorescent protein (GFP) (Figure 1A)
Summary
Increase in the copy number of genomic segments harboring oncogenes and therapy target genes (gene amplification) causes aggressive tumor phenotypes by promoting cancer progression and acquired therapy resistance [1,2,3,4]. Defining cellular processes and factors regulating gene amplification would help us to identify molecular targets for controlling aggressive tumors. Gene amplification is a spontaneous event and is initiated by endogenous DNA breaks. Endogenous breaks in cancer cells can arise from physiological stresses including replication stress, which directly involves DNA synthesis at a very vulnerable time [10,11]. Cellular processes/factors that protect stalled forks can be important regulators of gene amplification
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
