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- Title
Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.
- Authors
Akre, Monica K.; Starrett, Gabriel J.; Quist, Jelmar S.; Temiz, Nuri A.; Carpenter, Michael A.; Tutt, Andrew N. J.; Grigoriadis, Anita; Harris, Reuben S.
- 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.
- Subjects
CYTOSINE deaminase; GENETIC mutation; MOLECULAR cloning; NUCLEOTIDE sequencing; DOXYCYCLINE
- Publication
PLoS ONE, 2016, Vol 11, Issue 5, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0155391