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PLoS One. 2016 Jun 30;11(6):e0158581. doi: 10.1371/journal.pone.0158581. eCollection 2016.

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

Author information

1
Program in Genetics and Molecular Biology, Emory University, Atlanta, Georgia, United States of America.
2
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, United States of America.
3
Department of Microbiology, Oslo University Hospital and University of Oslo, Oslo, Norway.
4
Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.
5
Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
6
Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
7
Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia, United States of America.

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.

PMID:
27362559
PMCID:
PMC4928824
DOI:
10.1371/journal.pone.0158581
[Indexed for MEDLINE]
Free PMC Article

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