Hayashi Akiyo, Suenaga Naohiro, Shiomi Yasushi, Nishitani Hideo
Graduate School of Life Science, University of Hyogo, Kamigori, Ako-gun, Hyogo, 678-1297, Japan.
Methods Mol Biol. 2014;1170:367-82. doi: 10.1007/978-1-4939-0888-2_19.
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.
增殖细胞核抗原（PCNA）是一种DNA夹子，作为参与DNA复制相关过程诸多方面的各种蛋白质的平台作用于染色质。这些蛋白质中的大多数都有与PCNA相关的PCNA相互作用蛋白基序（PIP框）。最近的研究表明，PCNA作为一个媒人发挥着重要作用，将与PCNA相互作用的蛋白质连接到泛素连接酶CRL4（Cdt2）以进行降解。在哺乳动物细胞中，被CRL4（Cdt2）降解的蛋白质包括Cdt1、p21和Set8。这些CRL4（Cdt2）底物有一个PIP降解子，它由典型的PIP框序列和泛素化所需的额外保守氨基酸组成。当PCNA在S期开始时加载到染色质上时，这些蛋白质的降解就会被触发，这个过程对于防止DNA重新复制很重要。这些CRL4（Cdt2）底物在DNA损伤时也通过相同的机制被降解。在本章中，我们描述了几种方法来研究含PIP降解子的蛋白质如何以PCNA依赖的方式被降解。
