Mutation processes in 293-based clones overexpressing the DNA cytosine deaminase APOBEC3B

Monica K. Akre, Gabriel J. Starrett, Jelmar S. Quist, Nuri A. Temiz, Michael A. Carpenter, Andrew N J Tutt, Anita Grigoriadis, Reuben S. Harris

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

Original languageEnglish (US)
Article numbere0155391
JournalPloS one
Volume11
Issue number5
DOIs
StatePublished - May 1 2016

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cytosine deaminase
Cytosine Deaminase
Mutagenesis
Clone Cells
Cells
clones
mutation
Mutation
DNA
Genes
Doxycycline
Cytosine
Cell death
Microarrays
Aberrations
mutagenesis
Dilution
Genome
Repair
Deamination

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Mutation processes in 293-based clones overexpressing the DNA cytosine deaminase APOBEC3B. / Akre, Monica K.; Starrett, Gabriel J.; Quist, Jelmar S.; Temiz, Nuri A.; Carpenter, Michael A.; Tutt, Andrew N J; Grigoriadis, Anita; Harris, Reuben S.

In: PloS one, Vol. 11, No. 5, e0155391, 01.05.2016.

Research output: Contribution to journalArticle

Akre, MK, Starrett, GJ, Quist, JS, Temiz, NA, Carpenter, MA, Tutt, ANJ, Grigoriadis, A & Harris, RS 2016, 'Mutation processes in 293-based clones overexpressing the DNA cytosine deaminase APOBEC3B', PloS one, vol. 11, no. 5, e0155391. https://doi.org/10.1371/journal.pone.0155391
Akre, Monica K. ; Starrett, Gabriel J. ; Quist, Jelmar S. ; Temiz, Nuri A. ; Carpenter, Michael A. ; Tutt, Andrew N J ; Grigoriadis, Anita ; Harris, Reuben S. / Mutation processes in 293-based clones overexpressing the DNA cytosine deaminase APOBEC3B. In: PloS one. 2016 ; Vol. 11, No. 5.
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