Multiple end joining mechanisms repair a chromosomal DNA break in fission yeast

Non-homologous end joining (NHEJ) is an important mechanism for repairing DNA double-strand breaks (DSBs). The fission yeast Schizosaccharomyces pombe has a conserved set of NHEJ factors including Ku, DNA ligase IV, Xlf1, and Pol4. Their roles in chromosomal DSB repair have not been directly characterized before. Here we used HO endonuclease to create a specific chromosomal DSB in fission yeast and examined the imprecise end joining events allowing cells to survive the continuous expression of HO. Our analysis showed that cell survival was significantly reduced in mutants defective for Ku, ligase IV, or Xlf1. Using Sanger sequencing and Illumina sequencing, we have characterized in depth the repair junction sequences in HO survivors. In wild type cells the majority of repair events were one-nucleotide insertions dependent on Ku, ligase IV, and Pol4. Our data suggest that fission yeast Pol4 is important for gap filling during NHEJ repair and can extend primers in the absence of terminal base pairing with the templates. In Ku and ligase IV mutants, the survivors mainly resulted from two types of alternative end joining events: one used microhomology flanking the HO site to delete sequences of hundreds to thousands of base pairs, the other rejoined the break using the HO-generated overhangs but also introduced one- or two-nucleotide base substitutions. The chromosomal repair assay we describe here should provide a useful tool for further exploration of the end joining repair mechanisms in fission yeast.