Understanding DNA replication and DNA damage responses – which must proceed faithfully to prevent diseases such as cancer – requires the ability to monitor protein dynamics at active and damaged replication forks (sites of DNA duplication). Existing methods for studying replication fork machinery have been limited in resolution and sensitivity.
Now, David Cortez, Ingram Professor of Cancer Research, and colleagues have developed a procedure to isolate proteins on nascent DNA (iPOND). Their method, described in the March issue of Nature Protocols, can be applied to any proliferating cell type. It relies on the incorporation of a chemical “label” into newly synthesized DNA. The label can be modified by a chemistry reaction, and proteins linked to the DNA can be isolated and characterized.
The researchers have used the iPOND technique to identify proteins associated with active replication forks, to monitor changes in chromatin located various distances from the replication fork, and to detect proteins or modifications of proteins at damaged replication forks.
This research was supported by grants from the National Cancer Institute of the National Institutes of Health and by the Department of Defense Breast Cancer Research Program.
