title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells.
abstract: Mammalian cells are constantly and unavoidably exposed to DNA damage from endogenous and exogenous sources, frequently to the detriment of genomic integrity and biological function. Cells acquire a large number of chemically diverse lesions per day, and each can have a different genetic fate and biological consequences. However, our knowledge of how and when specific lesions are repaired or how they may compromise the fidelity of DNA replication or transcription and lead to deleterious biological endpoints in mammalian cells is limited. Studying individual lesions requires technically challenging approaches for the targeted introduction of defined lesions into relevant DNA sequences of interest. Here, we present a systematic analysis of factors influencing yield and an improved, efficient and reliable protocol for the production of mammalian expression phagemid vectors containing defined DNA base modifications in any sequence position of either complementary DNA strand. We applied our improved protocol to study the transcriptional mutagenesis-mediated phenotypic consequences of the common oxidative lesion 5-hydroxyuracil, placed in the G12 mutational hotspot of the KRAS oncogene. 5-OHU induced sustained oncogenic signaling in Neil1-/-Neil2-/- mouse cells. The resulting advance in technology will have broad applicability for investigation of single lesion DNA repair, mutagenesis, and DNA damage responses in mammalian cells.
authors.affiliation: Program in Genetics and Molecular Biology, Emory University, Atlanta, Georgia, United States of America.|Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Lucy
authors.initials: L
authors.lastname: Petrova
authors.affiliation: Department of Microbiology, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Christine
authors.initials: C
authors.lastname: Gran
authors.affiliation: Department of Microbiology, Oslo University Hospital and University of Oslo, Oslo, Norway.|Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Magnar
authors.initials: M
authors.lastname: Bjoras
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, United States of America.|Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia, United States of America.|Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, Georgia, United States of America.|Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Doetsch
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics*; D004249:DNA Damage; D004260:DNA Repair; D005822:Genetic Vectors; D016296:Mutagenesis; D009154:Mutation
chemical_list: D004247:DNA