Mutational signatures of non‐homologous and polymerase theta‐mediated end‐joining in embryonic stem cells

• Published in: The EMBO journal 2017-12, Vol.36 (24), p.3634-3649
• Main Authors: Schimmel, Joost, Kool, Hanneke, van Schendel, Robin, Tijsterman, Marcel
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; Cell survival; cNHEJ; Configurations; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; Deoxyribonucleic acid; DNA; DNA End-Joining Repair - genetics; DNA Polymerase beta - genetics; DNA Polymerase beta - metabolism; DNA Polymerase theta; DNA repair; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; double‐strand break repair; EMBO13; EMBO39; Embryo cells; embryonic stem cells; Embryonic Stem Cells - cytology; Embryonic Stem Cells - physiology; Embryos; Genomes; Homology; Hypoxanthine Phosphoribosyltransferase; Lethality; Mammals; Mice; Models, Genetic; Mutation; Non-homologous end joining; polymerase theta‐mediated end joining; Repair; Reproduction (copying); Signatures; Stem cell transplantation; Stem cells
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201796948

Cells employ potentially mutagenic DNA repair mechanisms to avoid the detrimental effects of chromosome breaks on cell survival. While classical non‐homologous end‐joining (cNHEJ) is largely error‐free, alternative end‐joining pathways have been described that are intrinsically mutagenic. Which end‐joining mechanisms operate in germ and embryonic cells and thus contribute to heritable mutations found in congenital diseases is, however, still largely elusive. Here, we determined the genetic requirements for the repair of CRISPR/Cas9‐induced chromosomal breaks of different configurations, and establish the mutational consequences. We find that cNHEJ and polymerase theta‐mediated end‐joining (TMEJ) act both parallel and redundant in mouse embryonic stem cells and account for virtually all end‐joining activity. Surprisingly, mutagenic repair by polymerase theta (Pol θ, encoded by the Polq gene) is most prevalent for blunt double‐strand breaks (DSBs), while cNHEJ dictates mutagenic repair of DSBs with protruding ends, in which the cNHEJ polymerases lambda and mu play minor roles. We conclude that cNHEJ‐dependent repair of DSBs with protruding ends can explain de novo formation of tandem duplications in mammalian genomes. Synopsis Embryonic stem cells utilize two distinct mutagenic end‐joining mechanisms to repair chromosomal breaks. The configuration of the break dictates repair‐pathway choice, resulting in different repair outcomes, which can explain mutational signatures observed in human disease and evolution. Theta‐mediated end‐joining (TMEJ) acts as a first line defense mechanism for chromosomal breaks in mouse embryonic stem cells. Mutational signatures obtained in various human disease can be explained by TMEJ‐dependent repair of chromosomal breaks. Classical non‐homologous end‐joining (cNHEJ) of chromosomal breaks with protruding ends can explain the formation of tandem duplications in mammalian genomes. The introduction of one single chromosomal break induces lethality in cells that lack both TMEJ and cNHEJ. Graphical Abstract DNA double‐strand break configuration dictates the choice between two distinct error‐prone repair pathways in mouse embryonic stem cells, resulting in different repair outcomes.