Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245, USA.
Mol Cell Biol. 2010 Sep;30(17):4120-33. doi: 10.1128/MCB.01135-09. Epub 2010 Jul 6.
During DNA polymerase switching, the Xenopus laevis Cip/Kip-type cyclin-dependent kinase inhibitor Xic1 associates with trimeric proliferating cell nuclear antigen (PCNA) and is recruited to chromatin, where it is ubiquitinated and degraded. In this study, we show that the predominant E3 for Xic1 in the egg is the Cul4-DDB1-XCdt2 (Xenopus Cdt2) (CRL4(Cdt2)) ubiquitin ligase. The addition of full-length XCdt2 to the Xenopus extract promotes Xic1 turnover, while the N-terminal domain of XCdt2 (residues 1 to 400) cannot promote Xic1 turnover, despite its ability to bind both Xic1 and DDB1. Further analysis demonstrated that XCdt2 binds directly to PCNA through its C-terminal domain (residues 401 to 710), indicating that this interaction is important for promoting Xic1 turnover. We also identify the cis-acting sequences required for Xic1 binding to Cdt2. Xic1 binds to Cdt2 through two domains (residues 161 to 170 and 179 to 190) directly flanking the Xic1 PCNA binding domain (PIP box) but does not require PIP box sequences (residues 171 to 178). Similarly, human p21 binds to human Cdt2 through residues 156 to 161, adjacent to the p21 PIP box. In addition, we identify five lysine residues (K180, K182, K183, K188, and K193) immediately downstream of the Xic1 PIP box and within the second Cdt2 binding domain as critical sites for Xic1 ubiquitination. Our studies suggest a model in which both the CRL4(Cdt2) E3- and PIP box-containing substrates, like Xic1, are recruited to chromatin through independent direct associations with PCNA.
在 DNA 聚合酶切换过程中,非洲爪蟾的 Cip/Kip 型细胞周期蛋白依赖性激酶抑制剂 Xic1 与三聚体增殖细胞核抗原 (PCNA) 结合,并被招募到染色质中,在那里它被泛素化和降解。在这项研究中,我们表明,在卵中 Xic1 的主要 E3 是 Cul4-DDB1-XCdt2(非洲爪蟾 Cdt2)(CRL4(Cdt2))泛素连接酶。全长 XCdt2 的添加到非洲爪蟾提取物中促进 Xic1 周转,而 XCdt2 的 N 端结构域(残基 1 至 400)不能促进 Xic1 周转,尽管它能够结合 Xic1 和 DDB1。进一步的分析表明,XCdt2 通过其 C 端结构域(残基 401 至 710)直接与 PCNA 结合,表明这种相互作用对于促进 Xic1 周转很重要。我们还确定了 Xic1 结合到 Cdt2 所需的顺式作用序列。Xic1 通过两个结构域(残基 161 至 170 和 179 至 190)直接与 Xic1 PCNA 结合域(PIP 盒)结合,直接与 Cdt2 结合,但不需要 PIP 盒序列(残基 171 至 178)。同样,人 p21 通过残基 156 至 161 与人 Cdt2 结合,紧邻 p21 PIP 盒。此外,我们确定了 PIP 盒下游的五个赖氨酸残基(K180、K182、K183、K188 和 K193)和第二个 Cdt2 结合结构域内作为 Xic1 泛素化的关键位点。我们的研究表明,CRL4(Cdt2)E3 和包含 PIP 盒的底物(如 Xic1)都通过与 PCNA 的独立直接结合被招募到染色质中。