Dna2 initiates resection at clean DNA double-strand breaks

Nucleic Acids Res. 2017 Nov 16;45(20):11766-11781. doi: 10.1093/nar/gkx830.


Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5' strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5' strand DNA ∼10-20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5' strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP-MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.

MeSH terms

  • Animals
  • Carrier Proteins / metabolism
  • DNA / genetics
  • DNA / metabolism*
  • DNA Breaks, Double-Stranded*
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA Repair Enzymes
  • DNA Repair*
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / metabolism
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Immunoblotting
  • MRE11 Homologue Protein / metabolism
  • Mutation
  • Protein Binding
  • Tumor Suppressor Proteins / metabolism
  • Xenopus Proteins / genetics
  • Xenopus Proteins / metabolism*
  • Xenopus laevis


  • Carrier Proteins
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Mre11 protein, Xenopus
  • NBN protein, Xenopus
  • RBBP8 protein, Xenopus
  • RPA1 protein, Xenopus
  • Rad50 protein, Xenopus
  • Tumor Suppressor Proteins
  • Xenopus Proteins
  • DNA
  • Endonucleases
  • MRE11 Homologue Protein
  • DNA Helicases
  • DNA2 protein, Xenopus
  • DNA Repair Enzymes