Differences in cell culture conditions can drastically affect stem cell physiology. the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and 84954-92-7 manufacture 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. Introduction 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 3 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 84954-92-7 manufacture repeats located near the DSB. SSA is always mutagenic, as the sequence between the repeats is deleted. SSA has different genetic requirements from Rabbit polyclonal to CapG 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 been utilized in studies of DNA repair as they can be propagated in culture and lack the genetic alterations present in cancer cells [11]. Previous studies that characterized DSB repair within both human embryonic stem cells (hESCs) and somatic stem cells have primarily utilized nonspecific clastogens, such as ionizing radiation (IR), to examine effects on survival and cell cycle arrest, as well as the efficiency of 84954-92-7 manufacture repair and the induction of gross chromosomal rearrangements [12], [13], [14], [15]. This approach has several shortcomings. First, even low doses of nonspecific clastogens.