In Vitro Study of Repair of DNA Interstrand Crosslinks

描述(申请人提供)：DNA交联型癌症化疗药物的疗效取决于许多因素，包括DNA被修复的能力。本项目主要研究人工合成的CNG-1，3链间DNA交联物的修复机制，作为临床相关产品的DNA交联剂的模型。该项目使用快速DNA修复试验来研究核苷酸切除修复(NER)对哺乳动物无细胞提取物中交联链的修复。这些实验是在吸附在96孔板上的DNA上进行的；修复是使用生物素/链霉亲和素-荧光团系统检测的。还研究了单个NER因子对交联底物的亲和力。通过凝胶迁移率改变分析或使用蛋白质特异性抗体对平板分析的改进来检测这些抗体。使用化学探针进行互补性结构研究，以确定链间交联物引起的构象或其他变化的量。该项目的结果将为DNA修复的机制和规避DNA交联剂的先天或后天抗性以及修复途径的潜在抑制剂的问题提供有价值的见解。

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.