A Xenopus Cell-Free System for Analysis of the Chfr Ubiquitin Ligase Involved in Control of Mitotic Entry

The checkpoint protein Chfr delays entry into mitosis in the presence of mitotic stress. We have analyzed the Chfr checkpoint pathway in the Xenopus cell-free system. We showed that Chfr is a ubiquitin ligase that targets polo-like kinase (Plk1) for degradation, leading to delayed activation of the Cdc25C phosphatase and prolonged inhibitory phosphorylation of Cdc2 at the G2/M transition. In this chapter, we will describe biochemical methods we developed to analyze the Chfr auto-ubiquitination activity and the ubiquitination of its substrate Plk1, as well as functional assays to investigate the Chfr pathway in Xenopus extracts.