title: The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability.
abstract: DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas, United States of America.
authors.forename: Sabine S
authors.initials: SS
authors.lastname: Lange
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas, United States of America.
authors.forename: Junya
authors.initials: J
authors.lastname: Tomida
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas, United States of America.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Boulware
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas, United States of America.
authors.forename: Sarita
authors.initials: S
authors.lastname: Bhetawal
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas, United States of America.|The Graduate School of Biomedical Sciences at Houston, Houston, Texas, United States of America.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Wood
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004622:Embryo, Mammalian; D047108:Embryonic Development / Q000235:genetics*; D055879:Gene Knock-In Techniques; D042822:Genomic Instability; D051379:Mice; D009154:Mutation; D000099270:Y-Family DNA Polymerases
chemical_list: D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C486862:REV3L protein, mouse