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.

在我们先前的研究中，我们解决了FAM111A丝氨酸蛋白酶域（SPD）的X射线晶体结构。我们的生化和结构研究表明，FAM111A SPD通过N末端的Alpha-1螺旋形成同型二聚体，在那里，通过疏水相互作用将两个亚基固定在一起。用天冬氨酸在二聚化界面上取代丝网残基在体外减少了二聚体的形成和蛋白酶活性，以及​​在细胞中保护复制叉免受topoisomerase抑制剂camptothecin诱导的DPC的能力。为了了解SPD的二聚化如何促进蛋白酶活性，我们解决了缺乏二聚体界面的一部分的SPD突变体的X射线晶体结构。有趣的是，不仅二聚化界面，而且氧孔（一种对蛋白酶活性至关重要的活性位点结构）在单体突变体中都是无序的。这些新数据揭示了FAM111A SPD的二聚化依赖性活性的机制，其中二聚化触发了一系列无序的转变，从而导致氧气孔的稳定。这些结构研究将有助于我们确定潜在的机制，这些机制可以调节在DPC处停滞的复制叉时FAM111A的蛋白酶活性，并促进FAM111A抑制剂的发展作为抗癌疗法的敏感性，包括拓扑异构酶抑制剂。