PCNA-dependent ubiquitination of Cdt1 and p21 in mammalian cells.

Methods Mol Biol

Graduate School of Life Science, University of Hyogo, Kamigori, Ako-gun, Hyogo, 678-1297, Japan.

Published: January 2015

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 CRL4(Cdt2) for their degradation. Proteins degraded by CRL4(Cdt2) include Cdt1, p21, and Set8 in mammalian cells. These CRL4(Cdt2) 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 CRL4(Cdt2) 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.

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http://dx.doi.org/10.1007/978-1-4939-0888-2_19DOI Listing

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