PCNA-Dependent Ubiquitination of Cdt1 and p21 in Mammalian Cells

PCNA is a DNA clamp, acting on chromatin as a platform for various proteins involved in many aspects of DNA replication-linked processes. Most of these proteins have the PCNA-interaction protein motif (PIP box) that associates with PCNA. Recent works show that PCNA plays an important role as a matchmaker, connecting PCNA-interacting proteins to the ubiquitin ligase CRL4Cdt2 for their degradation. Proteins degraded by CRL4Cdt2 include Cdt1, p21, and Set8 in mammalian cells. These CRL4Cdt2 substrates have a PIP degron that consists of the canonical PIP-box sequence and additional conserved amino acids required for ubiquitination. The degradation of these proteins is triggered when PCNA is loaded onto chromatin at the onset of S phase, and this process is important to prevent re-replication of DNA. These CRL4Cdt2 substrates are also degraded through the same mechanism in response to DNA damage. In this chapter, we describe several approaches to investigate how PIP degron-containing proteins are degraded in a PCNA-dependent manner.

Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news