DNA Replication and Repair

DNA复制与修复

• 批准号: 10926493
• 负责人: Yuichi Machida
• 金额: $ 148.6万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10926493
• 关键词: Active Sites; Aspartate; Biochemical; Camptothecin; Cells; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA replication fork; DNA-protein crosslink; Data; Development; Dimerization; Disease; Goals; Hydrophobic Interactions; In Vitro; Movement; Peptide Hydrolases; Pharmaceutical Preparations; Protease Domain; Proteins; Regulation; Roentgen Rays; Serine Protease; Structure; Therapeutic; Topoisomerase Inhibitors; Valine; anti-cancer therapeutic; cancer cell; crosslink; dimer; improved; inhibitor; monomer; mutant

In our previous study, we have solved X-ray crystal structures of the FAM111A serine protease domain (SPD). Our biochemical and structural studies suggested that FAM111A SPD forms a homodimer through the alpha-1 helix at the N-terminus, where two subunits are held together through hydrophobic interactions. Substitution of the valine residue on the dimerization interface with aspartate diminished dimer formation and protease activity of the SPD in vitro, as well as the ability to protect replication forks from DPCs induced by a topoisomerase inhibitor, camptothecin, in cells. To understand how dimerization of the SPD promotes the protease activity, we have solved X-ray crystal structures of an SPD mutant that lacks a part of the dimerization interface. Interestingly, not only the dimerization interface, but also the oxyanion hole, an active site structure critical for protease activity, is disordered in the monomeric mutants. These new data reveal a mechanism of the dimerization-dependent activity of the FAM111A SPD, in which dimerization triggers a cascade of disorder-to-order transitions that leads to the stabilization of the oxyanion hole. These structural studies will help us determine potential mechanisms that regulate the protease activity of FAM111A at replication forks stalled at DPCs and facilitate the development of FAM111A inhibitors as a sensitizers of anti-cancer therapeutics including topoisomerase inhibitors.

在我们之前的研究中，我们已经解决了FAM111A丝氨酸蛋白酶结构域(SPD)的X射线晶体结构。我们的生化和结构研究表明，FAM111ASPD通过N-末端的α-1螺旋形成同源二聚体，在那里两个亚基通过疏水相互作用连接在一起。用天冬氨酸取代二聚化界面上的Valine残基，在体外减少了SPD的二聚体形成和蛋白酶活性，以及在细胞内保护复制叉免受拓扑异构酶抑制剂喜树碱诱导的DPC的影响。为了了解SPD的二聚化如何促进蛋白酶活性，我们解决了一个SPD突变体的X射线晶体结构，该突变体缺乏部分二聚化界面。有趣的是，在单体突变体中，不仅二聚界面，而且氧阴离子空穴--一个对蛋白酶活性至关重要的活性部位结构--也是无序的。这些新数据揭示了FAM111ASPD的二聚依赖活性的机制，在该机制中，二聚触发了一系列无序到有序的跃迁，从而导致氧阴离子空穴的稳定。这些结构研究将有助于我们确定在DPC停滞不前的复制叉处调节FAM111a蛋白酶活性的潜在机制，并促进FAM111a抑制剂作为包括拓扑异构酶抑制剂在内的抗癌治疗的增敏剂的开发。