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

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.     

Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.