Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms, Herts, UK.
EMBO J. 2009 Dec 2;28(23):3657-66. doi: 10.1038/emboj.2009.303. Epub 2009 Oct 22.
Poly-ubiquitylation is a common post-translational modification that can impart various functions to a target protein. Several distinct mechanisms have been reported for the assembly of poly-ubiquitin chains, involving either stepwise transfer of ubiquitin monomers or attachment of a preformed poly-ubiquitin chain and requiring either a single pair of ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3), or alternatively combinations of different E2s and E3s. We have analysed the mechanism of poly-ubiquitylation of the replication clamp PCNA by two cooperating E2-E3 pairs, Rad6-Rad18 and Ubc13-Mms2-Rad5. We find that the two complexes act sequentially and independently in chain initiation and stepwise elongation, respectively. While loading of PCNA onto DNA is essential for recognition by Rad6-Rad18, chain extension by Ubc13-Mms2-Rad5 is only slightly enhanced by loading. Moreover, in contrast to initiation, chain extension is tolerant to variations in the attachment site of the proximal ubiquitin moiety. Our results provide information about a unique conjugation mechanism that appears to be specialised for a regulatable pattern of dual modification.
多聚泛素化是一种常见的翻译后修饰，可为靶蛋白赋予多种功能。现已报道了几种用于组装多泛素链的不同机制，涉及泛素单体的逐步转移或预形成的多泛素链的附着，并且需要单个泛素连接酶 (E2) 和泛素连接酶 (E3) 对，或者替代地由不同的 E2 和 E3 对组合。我们分析了由两个合作的 E2-E3 对 Rad6-Rad18 和 Ubc13-Mms2-Rad5 对复制夹子 PCNA 的多泛素化的机制。我们发现这两个复合物分别在链起始和逐步延伸中顺序和独立地起作用。虽然 PCNA 在 DNA 上的加载对于 Rad6-Rad18 的识别是必需的，但 Ubc13-Mms2-Rad5 的链延伸仅略有增强。此外，与起始相反，链延伸对近端泛素部分的附着位点的变化具有耐受性。我们的结果提供了有关独特的缀合机制的信息，该机制似乎专门用于可调节的双重修饰模式。
