In Vitro Study of Repair of DNA Interstrand Crosslinks

描述（由申请人提供）：DNA交联癌症化疗药物的疗效依赖于许多因素，包括DNA修复的能力。该项目的重点是合成CNG 1，3链间DNA交联的修复机制，作为处方DNA交联剂的临床相关产品的模型。该项目采用快速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的修复。这些实验在已经吸附到96孔板上的DNA上进行;使用生物素/链霉亲和素-荧光团系统检测修复。还研究了个别NER因子对交联基质的亲和力。这些通过电泳迁移率变动测定或使用蛋白质特异性抗体的平板测定的修改来检查。进行涉及使用化学探针的互补结构研究，以确定由链间交联诱导的构象或其他变化的量。这个项目的结果应该提供有价值的见解DNA修复的机制和方法规避先天或后天耐药的问题，DNA交联剂以及潜在的抑制剂的修复途径。

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.