DNA mediated chromatin pull-down for the study of chromatin replication

Sci Rep. 2011;1:95. doi: 10.1038/srep00095. Epub 2011 Sep 19.

Abstract

Chromatin replication involves duplicating DNA while maintaining epigenetic information. These processes are critical for genome stability and for preserving cell-type identity. Here we describe a simple experimental approach that allows chromatin to be captured and its content analysed after in vivo replication and labeling of DNA by cellular DNA polymerases. We show that this technique is highly specific and that proteins bound to the replicated DNA can be analyzed by both immunological techniques and large scale mass spectrometry. As proof of concept we have used this novel procedure to begin investigating the relationship between chromatin protein composition and the temporal programme of DNA replication in human cells. It is expected that this technique will become a widely used tool to address how chromatin proteins assemble onto newly replicated DNA after passage of a replication fork and how chromatin maturation is coupled to DNA synthesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatin / metabolism*
  • Chromatin Immunoprecipitation
  • DNA / metabolism*
  • DNA Replication
  • DNA-Directed DNA Polymerase / metabolism
  • HeLa Cells
  • Humans

Substances

  • Chromatin
  • DNA
  • DNA-Directed DNA Polymerase