ChIP Technique to Study Protein Dynamics at Defined DNA Double Strand Breaks.

Abstract

Information about the timing of appearance and composition of protein aggregates, termed foci, that arise in eukaryotic cells at sites of DNA double strand breaks (DSBs) has been mainly obtained through immunostaining and thus is limited by the resolution of light microscopy and the availability of appropriate antibodies. In this chapter, we describe a system using direct protein transduction of homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs, allowing for detection of target proteins using chromatin immunoprecipitation (ChIP), enabling molecular probing of the cellular response to a DNA DSB. Following the introduction of I-PpoI and generation of DSBs, genomic DNA and protein are cross-linked and analyzed by ChIP to determine the spatial distribution and temporal accumulation of specific proteins at the site of breaks. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of I-PpoI in live cells, facilitating comparisons across multiple cell lines. This system allows the direct detection of protein and chromatin dynamics at the site of the break, as well as timing and extent of DNA DSB repair in human cells.