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.

Fig. 1. Click chemistry addition of biotin tags to nascent DNA

EdU incorporated into nascent DNA is covalently tagged with biotin in the copper-catalyzed click reaction. Orange color represents the functional groups involved in the click chemistry reaction.

Fig. 2. Schematic overview of the iPOND procedure

The iPOND procedure consists of pulsing cells with EdU to label nascent DNA in vivo, formaldehyde crosslinking protein-DNA complexes, covalently tagging EdU-labeled DNA with biotin using click chemistry, lysing and sonicating cells, purifying the solubilized protein-DNA complexes, and eluting bound proteins for analysis by SDS-PAGE and immunoblotting or MS. Numbering corresponds to the procedure step number

Fig. 3. Schematic of experimental procedures used to identify replisome or DNA damage proteins and modifications at the replication fork

A. To identify replisome proteins, a pulse-chase variation of the iPOND protocol employs a thymidine chase to move the nascent, EdU-labeled DNA segment away from the replication fork. The chase sample provides a control to distinguish replisome components from general chromatin binding factors. B. To study proteins and modifications associated with damaged replication forks, an agent that stalls replication forks such as hydroxyurea is added following the EdU labeling period.

Fig. 4. Example of results obtainable with iPOND

Cells were pulsed with EdU for 10 minutes and then incubated with thymidine for 0, 10, or 30 minutes as indicated.. iPOND was performed as described in the protocol. Eluted proteins (lanes 5-8) were analyzed by SDS-PAGE followed by immunoblotting for the replication proteins PCNA, chromatin assembly factor 1 (CAF-1/p60), and histone H2B. As expected, proteins are detectable in every sample of the input (lanes 1-4). In the absence of click chemistry (No Clk, lane 5, negative control), no proteins are isolated from nascent DNA. PCNA and CAF-1 are enriched specifically at the replication fork (Click rxn, lane 6), but not on nascent DNA that is thymidine chased away from the replication fork (Click rxn, lanes 7,8). In contrast, a chromatin bound protein such as H2B is detectable both at the replication fork (Click rxn, lane 6) and in thymidine chased samples (Click rxn, lanes 7,8).