SUMO-BINDING MOTIFS MEDIATE THE RAD60-DEPENDENT RESPONSE

SUMO 结合基序调节 RAD60 依赖性反应

• 批准号: 7602145
• 负责人: MICHAEL N BODDY
• 金额: $ 0.62万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602145
• 关键词: Alanine; Binding; C-terminal; Cell Cycle Progression; Cells; Computer Retrieval of Information on Scientific Projects Database; Condition; DNA Repair; Defect; Ensure; FHA Domain; Fission Yeast; Funding; Genome; Grant; Human; In Vitro; Institution; Mediating; Mitosis; Mutation; Nuclear Protein; Nuclear Proteins; Phosphorylation; Phosphorylation Site; Physiological; Proteins; Research; Research Personnel; Resources; Source; Stress; United States National Institutes of Health; checkpoint kinase 2; helicase; in vivo; mutant; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In fission yeast, the replication checkpoint is enforced by the kinase Cds1 (human Chk2), which regulates both cell cycle progression and DNA repair factors to ensure that the genome is faithfully duplicated prior to mitosis. Cds1 contains a forkhead-associated domain that mediates its interaction with phosphorylated residues in target proteins. One target of Cds1 is the essential nuclear protein Rad60, which contains the unique structural feature of tandem SUMO homology domains at its C terminus. Hypomorphic mutants of Rad60 cause profound defects in DNA repair and replication stress tolerance. To explore the physiological significance of the Cds1-Rad60 interaction, we have examined the phosphorylation of Rad60 by Cds1 in vitro and the in vivo phosphorylation of Rad60 in response to replication blocks. We find that the N terminus but not the SUMO-like domain of Rad60 is phosphorylated in both conditions. Three important Rad60 phosphorylation sites were identified: Thr72, Ser32, and Ser34. Rad60 Thr72 mediates the Cds1-Rad60 interaction and is required for the Cds1-dependent phosphorylation of Rad60 in response to replication arrest. Phosphorylation of Rad60 Ser32 and Ser34 in a putative SUMO-binding motif is critical for the survival of replication stress. In addition, mutation of Rad60 Ser32 and Ser34 to alanine is lethal in cells deleted for the RecQ DNA helicase Rqh1. Finally, we find that Rad60 self-associates via its C-terminal SUMO-like domain and putative SUMO-binding motifs.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在分裂酵母中，复制检查点由激酶Cds 1（人Chk 2）强制执行，它调节细胞周期进程和DNA修复因子，以确保基因组在有丝分裂前忠实复制。cds 1包含一个叉头相关结构域，介导其与靶蛋白中磷酸化残基的相互作用。Cds 1的一个靶标是必需的核蛋白Rad 60，其在其C末端含有串联SUMO同源结构域的独特结构特征。Rad 60的亚纯型突变体在DNA修复和复制应激耐受性中引起严重缺陷。为了探索Cds 1-Rad 60相互作用的生理意义，我们研究了Cds 1在体外对Rad 60的磷酸化和在体内对Rad 60的磷酸化对复制阻断的反应。我们发现，在这两种条件下，N末端，但不是SUMO样结构域的Rad 60磷酸化。三个重要的Rad 60磷酸化位点被确定：Thr 72，Ser 32和Ser 34。Rad 60 Thr 72介导Cds 1-Rad 60相互作用，并且是响应于复制停滞的Rad 60的Cds 1依赖性磷酸化所必需的。Rad 60 Ser 32和Ser 34在一个假定的SUMO结合基序的磷酸化是复制应激的生存至关重要。此外，Rad 60 Ser 32和Ser 34突变为丙氨酸在缺失RecQ DNA解旋酶Rqh 1的细胞中是致命的。最后，我们发现Rad 60通过其C-末端SUMO样结构域和假定的SUMO结合基序进行自缔合。