DNA Replication and Repair

DNA复制与修复

• 批准号: 10702862
• 负责人: Yuichi Machida
• 金额: $ 118.11万
• 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/10702862
• 关键词: Aspartate; Biochemical; Camptothecin; Chemicals; Crystallization; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA replication fork; DNA-protein crosslink; Data; Dimerization; Enzymes; Goals; Hydrophobic Interactions; Movement; Peptide Hydrolases; Pharmaceutical Preparations; Protease Domain; Proteins; Regulation; Roentgen Rays; Serine Protease; Structure; Valine; cancer cell; crosslink; dimer; experimental study; improved; in vivo; inhibitor; mutant; prevent

We have solved X-ray crystal structures of the FAM111A serine protease domain (SPD). The structural information will help us determine potential mechanisms that regulate the protease activity of FAM111A. 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. We found that substitution of valine on the dimerization interface with aspartate disrupted dimer formation and diminished its protease activity. These data suggest that the dimerization of the enzyme domain is crucial for the activity of FAM111A. Supporting the importance of the dimer formation, our in vivo experiments suggested that the monomeric mutant was defective in preventing DPC accumulation and failed to promote DNA replication at DPCs induced by camptothecin. These results raise a new concept that chemicals that can block dimer formation could be used as an inhibitor of FAM111A.

我们已经解析了 FAM111A 丝氨酸蛋白酶结构域 (SPD) 的 X 射线晶体结构。 结构信息将帮助我们确定调节 FAM111A 蛋白酶活性的潜在机制。我们的生化和结构研究表明，FAM111A SPD 通过 N 末端的 α-1 螺旋形成同二聚体，其中两个亚基通过疏水相互作用结合在一起。我们发现二聚化界面上的缬氨酸被天冬氨酸取代会破坏二聚体的形成并降低其蛋白酶活性。这些数据表明酶结构域的二聚化对于 FAM111A 的活性至关重要。我们的体内实验表明，单体突变体在防止 DPC 积累方面存在缺陷，并且无法促进喜树碱诱导的 DPC 中的 DNA 复制，这支持了二聚体形成的重要性。这些结果提出了一个新概念，即可以阻止二聚体形成的化学物质可以用作 FAM111A 的抑制剂。