title: Analysis of protein dynamics at active, stalled, and collapsed replication forks.
abstract: Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks at high resolution. Using this methodology, we define the timing of histone deposition and chromatin maturation. Class 1 histone deacetylases are enriched at replisomes and remove predeposition marks on histone H4. Chromatin maturation continues even when decoupled from replisome movement. Furthermore, fork stalling causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to fork collapse and double-strand break formation. Finally, we demonstrate a switch in the DDR at persistently stalled forks that includes MRE11-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Bianca M
authors.initials: BM
authors.lastname: Sirbu
authors.forename: Frank B
authors.initials: FB
authors.lastname: Couch
authors.forename: Jordan T
authors.initials: JT
authors.lastname: Feigerle
authors.forename: Srividya
authors.initials: S
authors.lastname: Bhaskara
authors.forename: Scott W
authors.initials: SW
authors.lastname: Hiebert
authors.forename: David
authors.initials: D
authors.lastname: Cortez
mesh_terms: D002843:Chromatin / Q000378:metabolism; D057930:Click Chemistry / Q000379:methods; D004249:DNA Damage; D004261:DNA Replication / Q000502:physiology*; D057809:HEK293 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans
chemical_list: D002843:Chromatin; C468783:H2AX protein, human; D006657:Histones