Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

• Type of Material: Article
• Authors: Lucy Petrova, Emory University; Christine Gran, Oslo University Hospital; Magnar Bjoras, Oslo University Hospital; Paul Doetsch, Emory University
• Language: English
• Date: 2016-06-30
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2016 Petrova et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0158581
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 11
• Issue: 6
• Start Page: e0158581
• End Page: e0158581
• Grant/Funding Information: National Cancer Institute CA120288 to Paul W. Doetsch.; Funded by National Institutes of Health ES011163 CA120288.; National Institute of Environmental Health Sciences ES011163 to Paul W. Doetsch.
• Keywords: TRANSCRIPTIONAL MUTAGENESIS; DIHYDROURACIL; Science & Technology; STRANDED-DNA; Science & Technology - Other Topics; ENDONUCLEASE-III; BASE MODIFICATIONS; Multidisciplinary Sciences; N-GLYCOSYLASE; ESCHERICHIA-COLI; ASSAYS; OXIDIZED BASES; URACIL
• Research Categories: Health Sciences, Oncology; Biology, Genetics; Biology, Molecular

Abstract

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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 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.