Abstract
Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.
Highlights
The preservation of genomic integrity requires the recognition and repair of a vast array of DNA damage, including strand breaks and chemical base modifications
homologous recombination (HR) is generally considered to be a precise form of repair, because it can restore the original sequence if the sister chromatid or another identical sequence is used as a template [4]
We demonstrate that the phenotype of repair at a single double-strand break (DSB) differs across isogenic human embryonic stem cells (hESCs) based on either the site of the DSB within the genome or the stage of cellular differentiation
Summary
The preservation of genomic integrity requires the recognition and repair of a vast array of DNA damage, including strand breaks and chemical base modifications. DNA double-strand breaks (DSBs) are challenging to repair, as neither strand remains intact to template repair for the other. DSB repair in mammalian cells either utilizes a homologous template or involves nonhomologous end-joining (NHEJ). DSB repair that utilizes a homologous template can either involve homologous recombination (HR) or single-strand annealing (SSA) [3]. In both pathways, the DSB end is processed to a single-strand 39 tail. In HR, the single-strand tail undergoes RAD51-dependent invasion of a homologous duplex followed by template-dependent synthesis. HR is generally considered to be a precise form of repair, because it can restore the original sequence if the sister chromatid or another identical sequence is used as a template [4]. HR between homologous chromosomes can result in loss of heterozygosity
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