Isolation of Proteins on Nascent DNA in Hypoxia and Reoxygenation Conditions.
During S-phase both DNA replication and histone deposition must be co-ordinated at and around the replication fork. Replication stress can interfere with the fidelity of this process and can result in genomic instability. The study of proteins associated with DNA replication forks is important for a detailed understanding of DNA replication and chromatin assembly both under basal as well as replication stress conditions. iPOND (isolation of Proteins on Nascent DNA) allows the temporal study of proteins and protein modifications associated with replication forks in a variety of conditions, allowing the 'tracing' of protein association and histone deposition and maturation at active, stalled and damaged replication forks. Importantly, low oxygen (hypoxic) conditions, found in tumours, can result in replication stress. Here we describe the adaptation of the iPOND technique allowing the isolation of proteins and protein modifications specifically with replication forks undergoing hypoxia-induced replication stress. Furthermore, we describe the adaptation of this method for the study of factors associated with replication forks recovering from hypoxia-induced replication stress following periods of reoxygenation. These adaptations are important in order to study proteins associated with replication forks undergoing replication stress in physiologically relevant conditions.
CR-UK & MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK. molcina@stanford.edu.|Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, CA, 94305, USA. molcina@stanford.edu.
Monica M
MM
Olcina
Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, CA, 94305, USA.
Amato J
AJ
Giaccia
CR-UK & MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
Ester M
EM
Hammond
D000818:Animals; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D015687:Cell Hypoxia; D002460:Cell Line; D002463:Cell Membrane Permeability; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D005557:Formaldehyde / Q000378:metabolism; D006801:Humans; D010100:Oxygen / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification*
D003432:Cross-Linking Reagents; D011506:Proteins; D005557:Formaldehyde; D001710:Biotin; D004247:DNA; D010100:Oxygen
5-Ethynyl-2′-deoxyuridine (EdU); Chromatin; Click chemistry; DNA replication; Hypoxia; Reoxygenation; Replication stress; iPOND