Immunoprecipitation of p97 from X.laevis S-phase chromatin

Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process emerged: the replicative helicase at the heart of the replisome is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. To understand how p97 is targeted to the terminated replisome, we immunoprecipitated p97 from chromatin replicating in Xenopus laevis egg extract. We have found a p97 cofactor, Ubxn7, which can interact with active Cul2Lrr1 and with p97, facilitating efficient recognition and processing of terminated helicases ubiquitylated by Cul2.