Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells

PLoS Genet. 2010 Feb 26;6(2):e1000855. doi: 10.1371/journal.pgen.1000855.

Abstract

The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways-the main Ku heterodimer-dependent or "classic" NHEJ (C-NHEJ) pathway and an "alternative" NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PK(cs), XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PK(cs), XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PK(cs)-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cricetinae
  • DNA / genetics
  • DNA Breaks, Double-Stranded*
  • DNA Helicases / metabolism*
  • DNA Ligase ATP
  • DNA Ligases / deficiency
  • DNA Repair Enzymes / deficiency
  • DNA Repair Enzymes / metabolism
  • DNA Repair*
  • DNA-Activated Protein Kinase / deficiency
  • DNA-Activated Protein Kinase / metabolism
  • DNA-Binding Proteins / deficiency
  • DNA-Binding Proteins / metabolism
  • HCT116 Cells
  • Haploidy
  • Humans
  • Ku Autoantigen
  • Mutation / genetics
  • Nuclear Proteins / deficiency
  • Nuclear Proteins / metabolism
  • Plasmids / genetics
  • Recombination, Genetic*
  • Reproducibility of Results

Substances

  • DNA-Binding Proteins
  • NHEJ1 protein, human
  • Nuclear Proteins
  • DNA
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • DNA Helicases
  • XRCC5 protein, human
  • Ku Autoantigen
  • DNA Ligases
  • DNA Repair Enzymes
  • DNA Ligase ATP