The checkpoint protein Chfr delays entry into mitosis in the presence of mitotic stress. We have analyzed the Chfr checkpoint pathway in theXenopuscell-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 inXenopusextracts.
