title: Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA.
abstract: Understanding the processes of DNA replication, chromatin assembly and maturation, and the replication stress response requires the ability to monitor protein dynamics at active and damaged replication forks. Detecting protein accumulation at replication forks or damaged sites has primarily relied on immunofluorescence imaging, which is limited in resolution and antibody sensitivity. Here we describe a procedure to isolate proteins on nascent DNA (iPOND) that permits a high-resolution spatiotemporal analysis of proteins at replication forks or on chromatin following DNA replication in cultured cells. iPOND relies on labeling of nascent DNA with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). Biotin conjugation to EdU-labeled DNA using click chemistry facilitates a single-step streptavidin purification of proteins bound to the nascent DNA. iPOND permits an interrogation of any cellular process linked to DNA synthesis using a 3- to 4-d protocol.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Bianca M
authors.initials: BM
authors.lastname: Sirbu
authors.forename: Frank B
authors.initials: FB
authors.lastname: Couch
authors.forename: David
authors.initials: D
authors.lastname: Cortez
mesh_terms: D001710:Biotin / Q000737:chemistry; D002478:Cells, Cultured; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000737:chemistry; D015394:Molecular Structure; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011506:Proteins; D001710:Biotin; D004247:DNA; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine