Chromatin immunoprecipitation to detect DNA replication and repair factors

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1300; p. 169
• Main Authors: Gadaleta, Mariana C, Iwasaki, Osamu, Noguchi, Chiaki, Noma, Ken-Ichi, Noguchi, Eishi
• Format: Journal Article
• Language: English
• Published: United States 2015
• Subjects: Antibodies - pharmacology; Cell Cycle; Cell Extracts; Chromatin Immunoprecipitation - methods; Cross-Linking Reagents - pharmacology; DNA Repair; DNA Replication; Genomic Instability; Real-Time Polymerase Chain Reaction; Schizosaccharomyces - drug effects; Schizosaccharomyces - genetics; Schizosaccharomyces pombe Proteins - metabolism
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-2596-4_12

DNA replication is tightly coupled with DNA repair processes in order to preserve genomic integrity. During DNA replication, the replication fork encounters a variety of obstacles including DNA damage/adducts, secondary structures, and programmed fork-blocking sites, which are all difficult to replicate. The replication fork also collides with the transcription machinery, which shares the template DNA with the replisome complex. Under these conditions, replication forks stall, causing replication stress and/or fork collapse, ultimately leading to genomic instability. The mechanisms to overcome these replication problems remain elusive. Therefore, it is important to investigate how DNA repair and replication factors are recruited and coordinated at chromosomal regions that are difficult to replicate. In this chapter, we describe a chromatin immunoprecipitation method to locate proteins required for DNA repair during DNA replication in the fission yeast Schizosaccharomyces pombe. This method can also easily be adapted to study replisome components or chromatin-associated factors.