DNA mediated chromatin pull-down: a novel method for the study of chromatin replication

Abstract

Chromatin replication involves duplicating DNA while maintaining epigenetic information. These processes are critical for genome stability and for preserving cell-type identity. To investigate how these processes are regulated and coordinated, it is important to determine the identity of the proteins that are either stably or transiently associated with newly synthesised DNA. We have devised an experimental procedure that allows newly synthesised 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 analysed by both immunological techniques and large scale mass spectrometry. 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. We have focused on identifying the protein component of newly synthesised chromatin and in assessing qualitative and quantitative changes in this protein fraction caused by the normal temporal program of DNA replication as well as by perturbation of the DNA replication machinery. It is expected that this technique will become a leading methodology for the study of chromatin editing and maturation and for investigating how these processes are linked to the duplication of DNA.