Chromatin immunoprecipitation to detect DNA replication and repair factors.
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.
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 245 N 15th Street, Philadelphia, PA, 19102, USA.
Mariana C
MC
Gadaleta
Osamu
O
Iwasaki
Chiaki
C
Noguchi
Ken-Ichi
K
Noma
Eishi
E
Noguchi
D000906:Antibodies / Q000494:pharmacology; D002453:Cell Cycle; D002457:Cell Extracts; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004260:DNA Repair; D004261:DNA Replication; D042822:Genomic Instability; D060888:Real-Time Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*
D000906:Antibodies; D002457:Cell Extracts; D003432:Cross-Linking Reagents; D029702:Schizosaccharomyces pombe Proteins