A role for human homologous recombination factors in suppressing microhomology-mediated end joining

Nucleic Acids Res. 2016 Jul 8;44(12):5743-57. doi: 10.1093/nar/gkw326. Epub 2016 Apr 29.

Abstract

DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.

MeSH terms

  • BRCA1 Protein / antagonists & inhibitors
  • BRCA1 Protein / genetics*
  • BRCA1 Protein / metabolism
  • BRCA2 Protein / antagonists & inhibitors
  • BRCA2 Protein / genetics*
  • BRCA2 Protein / metabolism
  • Base Sequence
  • Biological Assay
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Line, Tumor
  • DNA / metabolism*
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Endodeoxyribonucleases
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Humans
  • MRE11 Homologue Protein
  • Mutation
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Osteoblasts / cytology
  • Osteoblasts / metabolism
  • Poly(ADP-ribose) Polymerases / genetics
  • Poly(ADP-ribose) Polymerases / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Recombinational DNA Repair*
  • Replication Protein A / antagonists & inhibitors
  • Replication Protein A / genetics*
  • Replication Protein A / metabolism
  • Sequence Alignment
  • Sequence Homology, Nucleic Acid

Substances

  • BRCA1 Protein
  • BRCA1 protein, human
  • BRCA2 Protein
  • BRCA2 protein, human
  • Carrier Proteins
  • DNA-Binding Proteins
  • MRE11 protein, human
  • Nuclear Proteins
  • RNA, Small Interfering
  • RPA1 protein, human
  • Replication Protein A
  • DNA
  • Poly(ADP-ribose) Polymerases
  • DNA polymerase theta
  • DNA-Directed DNA Polymerase
  • Endodeoxyribonucleases
  • MRE11 Homologue Protein
  • RBBP8 protein, human