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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
Mdm2 promotes genetic instability and transformation independent of p53
Alyssa Bouska1, Tamara Lushnikova, Silvia Plaza
1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Abstract:
Mdm2, a regulator of the tumor suppressor p53, is frequently overexpressed in human malignancies. Mdm2 also has unresolved, p53-independent functions that contribute to tumorigenesis. Here, we show that increased Mdm2 expression induced chromosome/chromatid breaks and delayed DNA double-strand break repair in cells lacking p53 but not in cells with a mutant form of Nbs1, a component of the Mre11/Rad50/Nbs1 DNA repair complex. A 31-amino-acid region of Mdm2 was necessary for binding to Nbs1. Mutation of conserved amino acids in the Nbs1 binding domain of Mdm2 inhibited Mdm2-Nbs1 association and prevented Mdm2 from delaying phosphorylation of H2AX and ATM-S/TQ sites, repair of DNA breaks, and resolution of DNA damage foci. Similarly, the mutation of eight amino acids in the Mdm2 binding domain of Nbs1 inhibited Mdm2-Nbs1 interaction and blocked the ability of Mdm2 to delay DNA break repair. Both Nbs1 and ATM, but not the ubiquitin ligase activity of Mdm2, were necessary to inhibit DNA break repair. Only Mdm2 with an intact Nbs1 binding domain was able to increase the frequency of chromosome/chromatid breaks and the transformation efficiency of cells lacking p53. Therefore, the interaction of Mdm2 with Nbs1 inhibited DNA break repair, leading to chromosome instability and subsequent transformation that was independent of p53.
Insights
Mdm2 protein overexpression causes DNA breaks and delays repair in p53-deficient cells by interacting with Nbs1, a DNA repair protein. This interaction promotes chromosome instability and cell transformation independently of p53.
Area of Science:
- Molecular biology
- Cancer research
- DNA repair mechanisms
Background:
- Mdm2 is a key regulator of the tumor suppressor p53 and is often overexpressed in cancers.
- Mdm2 possesses p53-independent functions crucial for tumor development.
Purpose of the Study:
- To investigate the p53-independent role of Mdm2 in DNA damage and repair.
- To elucidate the mechanism by which Mdm2 influences DNA double-strand break repair and genomic instability.
Main Methods:
- Utilized cell lines with and without functional p53.
- Investigated the interaction between Mdm2 and Nbs1 using mutation analysis.
- Assessed DNA double-strand break repair, H2AX phosphorylation, and DNA damage foci resolution.
- Evaluated chromosome/chromatid breaks and cell transformation efficiency.
Main Results:
- Mdm2 overexpression delayed DNA double-strand break repair and increased chromosome breaks in p53-deficient cells.
- A specific Mdm2 region (31 amino acids) was identified as essential for Nbs1 binding.
- Disruption of the Mdm2-Nbs1 interaction abrogated Mdm2's effects on DNA repair and genomic stability.
- Nbs1 and ATM, but not Mdm2's ligase activity, were required for inhibiting DNA break repair.
Conclusions:
- Mdm2 interacts with Nbs1 to inhibit DNA double-strand break repair, leading to chromosome instability and transformation in a p53-independent manner.
- The Mdm2-Nbs1 interaction represents a novel mechanism contributing to tumorigenesis.
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