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Architecture and assembly of Poly-SUMO chains on PCNA in Saccharomyces cerevisiae

机译：酿酒酵母PCNA上Poly-SUMO链的结构和组装

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摘要

Posttranslational modifications of proliferating cell nuclear antigen (PCNA), the eukaryotic processivity clamp for DNA polymerases, regulate the pathways by which replication problems are resolved. In the budding yeast Saccharomyces cerevisiae, ubiquitylation of PCNA in response to DNA damage facilitates the replicative bypass of lesions, whereas conjugation of the ubiquitin-related modifier (SUMO) prevents unscheduled crossover events during S phase. We have analyzed the SUMO modification pattern of budding yeast PCNA in vivo and in vitro and found that most aspects of our in vitro sumoylation reactions reflect the situation under physiological conditions. We show that two oligomeric SUMO chains of two to three moieties each, linked via internal sumoylation consensus motifs within the SUMO sequence, are assembled on PCNA. The SUMO-specific ligase Siz1 both stimulates the overall efficiency of sumoylation and selects the modification site on PCNA. Furthermore, ubiquitin and SUMO chains are assembled independently, and we found evidence that both modifiers can coexist in vivo on a common PCNA subunit. Our results demonstrate for the first time the in vivo assembly of polymeric SUMO chains of defined linkage on a physiological substrate in yeast, but they also indicate that SUMO-SUMO polymers are dispensable for PCNA(SUMO) function in replication and recombination. (C) 2007 Elsevier Ltd. All rights reserved.
机译：翻译后修饰的增殖细胞核抗原（PCNA）是DNA聚合酶的真核持续性钳位，它调节复制问题解决的途径。在萌芽的酿酒酵母中，响应DNA损伤的PCNA泛素化促进了损伤的复制旁路，而泛素相关修饰剂（SUMO）的结合可防止S期发生计划外的交叉事件。我们已经分析了发芽的酵母PCNA在体内和体外的SUMO修饰模式，发现我们体外磺酰化反应的大多数方面都反映了生理条件下的情况。我们显示，在PCNA上组装了两个低聚的SUMO链，每个链有2至3个部分，通过SUMO序列内的内部sumoylation共有基序相连。 SUMO特异的连接酶Siz1既可以提高总磺酰化效率，又可以选择PCNA上的修饰位点。此外，泛素和SUMO链是独立组装的，我们发现证据表明这两种修饰剂可以在体内共同存在于一个常见的PCNA亚基上。我们的结果首次证明了在酵母的生理底物上具有确定连接键的聚合SUMO链的体内组装，但它们还表明SUMO-SUMO聚合物对于PCNA（SUMO）在复制和重组中的功能是必不可少的。 （C）2007 Elsevier Ltd.保留所有权利。

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