In Vitro Study of Repair of DNA Interstrand Crosslinks

DNA 链间交联修复的体外研究

• 批准号: 6883630
• 负责人: TRACEY McGregor Mason
• 金额: $ 5.23万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-24 至 2007-08-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6883630
• 关键词: DNA repair; Escherichia coli; alkyl group; antibody; antineoplastics; binding proteins; biophysics; biotin; cell free system; circular dichroism; conformation; crosslink; fluorescent dye /probe; gel mobility shift assay; intermolecular interaction; molecular probes; molecular shape; neoplasm /cancer pharmacology; nuclear magnetic resonance spectroscopy; nucleic acid structure; oligonucleotides; plasmids; postdoctoral investigator; protein purification; transfection /expression vector

DESCRIPTION (provided by applicant): The efficacy of DNA crosslinking cancer chemotherapeutics relies on many factors including the ability for the DNA to be repaired. This project focuses on the mechanism of repair of synthetic CNG 1,3 interstrand DNA crosslinks as a model for clinically relevant products of prescribed DNA crosslinking agents. The project employs the use of a fast DNA repair assay to study the repair of the crosslinks in mammalian cell-free extracts by Nucleotide Excision Repair (NER). These experiments are conducted on DNA that has been adsorbed onto a 96 well plate; repair is detected using a biotin/streptavidin-fluorophore system. Also studied is the affinity of individual NER factors for the crosslinked substrate. These are examined by electrophoretic mobility shift assays or a modification of the plate assay using protein-specific antibodies. Complementary structural studies involving the use of chemical probes are conducted to determine the amount of conformational or other changes induced by an interstrand crosslink. The results of this project should provide valuable insights into the mechanism of DNA repair and ways of circumventing the problem of innate or acquired resistance to DNA crosslinking agents as well as potential inhibitors to pathways of repair.

描述（由申请人提供）：DNA交联的癌症化学治疗剂的功效取决于许多因素，包括修复DNA的能力。该项目侧重于合成CNG 1,3链间DNA交联的修复机理，作为规定DNA交联剂临床相关产品的模型。该项目采用快速DNA修复测定法研究通过核苷酸切除修复（NER）研究哺乳动物无细胞提取物中的交联的修复。这些实验是在吸附在96井板上的DNA上进行的。使用生物素/链霉亲和素氟化器系统检测维修。还研究了单个NER因子对交联底物的亲和力。通过电泳迁移率转移测定法或使用蛋白质特异性抗体对板测定法进行了检查。进行了涉及化学探针的互补结构研究，以确定构象的量或链间交叉链接引起的其他变化的量。该项目的结果应提供有关DNA修复机制的有价值的见解，以及避免对DNA交联药物的先天或获得性抗性问题的方法，以及对修复途径的潜在抑制剂。