title: Multiple end joining mechanisms repair a chromosomal DNA break in fission yeast.
abstract: 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.
authors.affiliation: National Institute of Biological Sciences, 7 Science Park Road, Zhongguancun Life Science Park, Beijing, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.forename: Kun
authors.initials: K
authors.lastname: Dou
authors.forename: Mei-Jun
authors.initials: MJ
authors.lastname: Zhang
authors.forename: Fang
authors.initials: F
authors.lastname: Suo
authors.forename: Li-Lin
authors.initials: LL
authors.lastname: Du
mesh_terms: D001483:Base Sequence; D019457:Chromosome Breakage; D002875:Chromosomes / Q000235:genetics* / Q000378:metabolism; D059766:DNA End-Joining Repair; D011088:DNA Ligases / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D009154:Mutation; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029702:Schizosaccharomyces pombe Proteins; C519230:XLF1 protein, S pombe; C503462:POL4 protein, S pombe; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases