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Phenotypic characterization of missense polymerase-δ mutations using an inducible protein-replacement system


Ghodgaonkar, Medini Manohar; Kehl, Patrick; Ventura, Ilenia; Hu, Liyan; Bignami, Margherita; Jiricny, Josef (2014). Phenotypic characterization of missense polymerase-δ mutations using an inducible protein-replacement system. Nature Communications, 5:4990.

Abstract

Next-generation sequencing has revolutionized the search for disease-causing genetic alterations. Unfortunately, the task of distinguishing the handful of causative mutations from rare variants remains daunting. We now describe an assay that permits the analysis of all types of mutations in any gene of choice through the generation of stable human cell lines, in which the endogenous protein has been inducibly replaced with its genetic variant. Here we studied the phenotype of variants of the essential replicative polymerase-δ carrying missense mutations in its active site, similar to those recently identified in familial colon cancer patients. We show that expression of the mutants but not the wild-type protein endows the engineered cells with a mutator phenotype and that the mutations affect the fidelity and/or the exonuclease activity of the isolated enzyme in vitro. This proof-of-principle study demonstrates the general applicability of this experimental approach in the study of genotype-phenotype correlations.

Abstract

Next-generation sequencing has revolutionized the search for disease-causing genetic alterations. Unfortunately, the task of distinguishing the handful of causative mutations from rare variants remains daunting. We now describe an assay that permits the analysis of all types of mutations in any gene of choice through the generation of stable human cell lines, in which the endogenous protein has been inducibly replaced with its genetic variant. Here we studied the phenotype of variants of the essential replicative polymerase-δ carrying missense mutations in its active site, similar to those recently identified in familial colon cancer patients. We show that expression of the mutants but not the wild-type protein endows the engineered cells with a mutator phenotype and that the mutations affect the fidelity and/or the exonuclease activity of the isolated enzyme in vitro. This proof-of-principle study demonstrates the general applicability of this experimental approach in the study of genotype-phenotype correlations.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2014
Deposited On:10 Feb 2015 15:17
Last Modified:05 Apr 2016 18:49
Publisher:Nature Publishing Group
ISSN:2041-1723
Publisher DOI:https://doi.org/10.1038/ncomms5990
PubMed ID:25241845

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