A rapid and efficient method to purify proteins at replication forks under native conditions

Biotechniques. 2013 Oct;55(4):204-6. doi: 10.2144/000114089.

Abstract

Tools for studying replication fork dynamics are critical for dissecting the mechanisms of DNA replication, DNA repair, histone deposition, and epigenetic memory. Isolation of protein on nascent DNA (iPOND) is an elegant method for purifying replication fork proteins. Here, we present accelerated native iPOND (aniPOND), a simplification of the iPOND procedure with improved protein yield. Cell membrane lysis and nuclei harvesting are combined in one step to reduce washes and minimize sample loss. A mild nuclei lysis protocol is then used to better preserve DNA-protein complexes. aniPOND is faster than iPOND, avoids formaldehyde cross-linking, and improves protein yield 5- and 20-fold for the CAF1-complex or PCNA respectively. Moreover, using aniPOND, but not iPOND, we could detect the polycomb repressive complex 2 (PRC2) components SUZ12, EZH2, and RBBP4 at replication forks. This faster, higher-yield method will facilitate MS analysis of replication fork complexes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / genetics
  • Chromatin / isolation & purification
  • DNA Repair / genetics
  • DNA Replication / genetics*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / isolation & purification*
  • Histones / genetics
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Proteins / genetics
  • Proteins / isolation & purification*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • CNOT8 protein, human
  • Chromatin
  • DNA-Binding Proteins
  • Histones
  • Proliferating Cell Nuclear Antigen
  • Proteins
  • Transcription Factors