Abstract
DNA replication and repair are critical processes for all living organisms to ensure faithful duplication and transmission of genetic information. Flap endonuclease 1 (Fen1), a structure-specific nuclease, plays an important role in multiple DNA metabolic pathways and maintenance of genome stability. Human FEN1 mutations that impair its exonuclease activity have been linked to cancer development. FEN1 interacts with multiple proteins, including proliferation cell nuclear antigen (PCNA), to form various functional complexes. Interactions with these proteins are considered to be the key molecular mechanisms mediating FEN1's key biological functions. The current challenge is to experimentally demonstrate the biological consequence of a specific interaction without compromising other functions of a desired protein. To address this issue, we established a mutant mouse model harboring a FEN1 point mutation (F343A/F344A, FFAA), which specifically abolishes the FEN1/PCNA interaction. We show that the FFAA mutation causes defects in RNA primer removal and long-patch base excision repair, even in the heterozygous state, resulting in numerous DNA breaks. These breaks activate the G2/M checkpoint protein, Chk1, and induce near-tetraploid aneuploidy, commonly observed in human cancer, consequently elevating the transformation frequency. Consistent with this, inhibition of aneuploidy formation by a Chk1 inhibitor significantly suppressed the cellular transformation. WT/FFAA FEN1 mutant mice develop aneuploidy-associated cancer at a high frequency. Thus, this study establishes an exemplary case for investigating the biological significance of protein-protein interactions by knock-in of a point mutation rather than knock-out of a whole gene.
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Acknowledgements
We thank V Bedell, D Bobadilla, J Collins, and M Slovak at the cytogenetic core-facility of COH for technical assistance with the karyotype analysis of mouse MEF cells and FISH detection of aneuploid cells in paraffin-embedded tissue sections of normal and lung tumor tissues. We thank QM Chen (University of Arizona) for critical review of the manuscript and SR da Costa (City of Hope) for editorial assistance. This work was supported by NIH grants R01 CA085344 and R01 CA073764 to BHS and in part by R01 CA081063 to SM and R01 CA084469 to GPP and by the Lung Cancer Program of City of Hope's Comprehensive Cancer Center.
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Supplementary information, Figure S1
Levels of cytoplasmic and nuclear FEN1 were similar in WT, WT/FFAA, and FFAA/FFAA MEF cells. (PDF 43 kb)
Supplementary information, Figure S2
FFAA nuclease extracts (NEs) displayed similar flap cleavage activities to the WT NEs. (PDF 68 kb)
Supplementary information, Figure S3
FFAA nuclease extracts (NEs) had similar ligation activities to the WT NEs on the nicked DNA substrates. (PDF 86 kb)
Supplementary information, Figure S4
Purified recombinant FFAA FEN1 proteins have similar FEN1 activity as the WT FEN1. (PDF 78 kb)
Supplementary information, Figure S5
ATR was activated in the FFAA cells spontaneously or in response to treatments with H2O2. (PDF 54 kb)
Supplementary information, Figure S6
The FFAA mutation induced cell cycle arrest at the prophase. (PDF 95 kb)
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Zheng, L., Dai, H., Hegde, M. et al. Fen1 mutations that specifically disrupt its interaction with PCNA cause aneuploidy-associated cancer. Cell Res 21, 1052–1067 (2011). https://doi.org/10.1038/cr.2011.35
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DOI: https://doi.org/10.1038/cr.2011.35
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