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.

Transcriptional mutagenesis reduces splicing fidelity in mammalian cells

“…All of the relatively few investigations that have examined the biological effects of lesion-induced transcriptional errors in mammalian systems to date reveal that TM can cause phenotypic changes. The TM caused by the presence of 8-oxoG or 5-hydroxyuracil at specific codons of HRAS or KRAS , respectively, in MEFs lacking OGG1 or NEIL1/2 increases the expression levels of mutated and constitutively active Ras proteins, giving rise to sustained downstream oncogenic signaling ( 21 , 50 ). Similarly, the presence of 8-oxoG on the transcribed strand of a gene encoding luciferase in MEFs deficient for OGG1 impaired the function of this protein due to TM ( 23 ).…”

Easily-controllable, helper phage-free single-stranded phagemid production system

“…To prepare ss phagemid DNAs, many researchers including us have utilized methods in which an F′ Escherichia coli strain with a phagemid containing the M13 or f1 origin of replication ( ori ) is infected with a helper phage [ 27 , 28 ]. However, the yields of ss DNA are poorly reproducible, and various conditions, such as titers of helper phages, multiplicities of infection (MOI), and densities of E. coli at the time of infection, must be controlled to consistently obtain sufficient quantities of ss phagemids in the conventional method [ 29 ]. In addition, the ss DNA production may be lower, and contaminated with the helper phage-derived ss DNAs, depending on the constitutions of phagemids.…”