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

Lucy Petrova,Magnar Bjoras,Paul W Doetsch,Christine Gran,Michael R Volkert

doi:10.1371/journal.pone.0158581

Lucy Petrova, Magnar Bjoras + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0158581

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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.

Highlights

Mammalian cells are continuously exposed to DNA damage, which can be detrimental to health and is associated with cancer, neurodegenerative disease and aging [1,2,3]
In order to produce phagemid single-stranded DNA, we infected log-phase DH12S E. coli cultures with the M13KO7 derivative of the M13 phage that preferentially packages phagemid ssDNA containing the f1 origin of replication, rather than its own genome, which it packages in the absence of phagemid [21]
We found that the DH12S E. coli culture density at the time of M13KO7 infection is a reliable predictor of ssDNA yield. ssDNA yield increases as optical density at 600 nm (OD600) increases, before plateauing at OD600 of approximately 0.6, yielding a several-fold increase compared to the ssDNA yields at OD600 of approximately 0.05 or 0.1 (Fig 1B and 1C)

Summary

Introduction

Mammalian cells are continuously exposed to DNA damage, which can be detrimental to health and is associated with cancer, neurodegenerative disease and aging [1,2,3]. DNA and RNA polymerase bypass efficiencies, lesion stability and replicative or transcriptional mutagenicity, as well as the type of mutation introduced when mutagenesis occurs differ for each specific lesion, and different lesions are recognized and repaired by different components of the DNA repair pathways. All of these factors, as well as the specific sequence position in which a lesion occurs, can determine biological outcomes. A highly mutagenic lesion occurring in an oncogene mutational hotspot and evading DNA repair is likely to result in detrimental biological consequences, in contrast to one that is rarely mutagenic or quickly repaired

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