Repair at Single Targeted DNA Double-Strand Breaks in Pluripotent and Differentiated Human Cells
Citation: Fung H, Weinstock DM (
Repair at Single Targeted DNA Double-Strand Breaks in Pluripotent and Differentiated Human Cells
Hua Fung 0
David M. Weinstock 0
Martin G. Marinus, University of Massachusetts Medical School, United States of America
0 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School , Boston, Massachusetts , United States of America
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.
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Funding: This work was supported by a Seed Grant from the Harvard Stem Cell Institute (http://www.hsci.harvard.edu) and a Career Award in the Biomedical
Sciences from the Burroughs Wellcome Fund (www.BWFund.org). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
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 particularly 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). The classical pathway of
NHEJ, which is essential for lymphocyte antigen receptor
rearrangements and ionizing radiation resistance, is mediated by
the DNA end-binding heterodimer KU70/KU80, the kinase
DNA-PKcs, the XRCC4/XLF/LIG4 ligase complex, and the
endonuclease Artemis [1,2].
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 can be mutagenic if the template is similar but
not identical to the broken sequence. For example, HR between
homologous chromosomes can result in loss of heterozygosity.
SSA, in contrast with HR, involves the annealing of sequence
repeats located near the DSB. SSA is always mutagenic, as the
sequence between the repeats is deleted. SSA has different genetic
requirements from HR and does not involve strand invasion [5].
The balance between DSB repair pathways is a key determinant
of repair precision, and appears to differ between cell types and
during different phases of the cell cycle [6]. HR is most active
during the late S and G2 phases, when the sister chromatid is
available to template repair. NHEJ predominates in G0 and G1,
when HR could promote loss of heterozygosity, but remains active
throughout the cell cycle [2]. At least to some extent, the pathways
are competitive. For example, loss of classical NHEJ factors
promotes HR at an endonuclease-mediated DSB [7]. Similarly,
loss of NHEJ proteins can restore homologous recombination and
mitomycin C resistance in cells lacking HR factors [8,9,10].
Stem cells, including embryonic stem cells, have bee (...truncated)