Identification of the Xenopus DNA2 protein as a major nuclease for the 5'->3' strand-specific processing of DNA ends

Nucleic Acids Res. 2008 Nov;36(19):6091-100. doi: 10.1093/nar/gkn616. Epub 2008 Sep 27.

Abstract

The first step of homology-dependent DNA double-strand break (DSB) repair is the 5' strand-specific processing of DNA ends to generate 3' single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5' strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5'-->3' degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5'-->3' degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA / chemistry
  • DNA / metabolism
  • DNA Breaks, Double-Stranded
  • DNA Helicases / antagonists & inhibitors
  • DNA Helicases / isolation & purification
  • DNA Helicases / metabolism*
  • DNA Repair*
  • DNA, Single-Stranded / metabolism*
  • Endonucleases / antagonists & inhibitors
  • Endonucleases / isolation & purification
  • Endonucleases / metabolism*
  • Exodeoxyribonucleases / antagonists & inhibitors
  • Exodeoxyribonucleases / isolation & purification
  • Exodeoxyribonucleases / metabolism*
  • Xenopus Proteins / antagonists & inhibitors
  • Xenopus Proteins / isolation & purification
  • Xenopus Proteins / metabolism*
  • Xenopus laevis

Substances

  • DNA, Single-Stranded
  • Xenopus Proteins
  • DNA
  • Endonucleases
  • Exodeoxyribonucleases
  • DNA Helicases
  • DNA2 protein, Xenopus