Protein Purification and DNA Substrates Core

复杂的DNA损伤，如DNA链间交叉连接和三螺旋结构，在功能上对哺乳动物基因组造成破坏，并对DNA修复系统构成独特的挑战。复杂损伤的修复很可能涉及不同DNA修复途径的协同作用，如同源重组修复、切除修复和错配修复。我们提议的计划项目将通过分子生物学、遗传学和生化方法对复杂DNA损伤的处理进行全面的调查。蛋白质因子和DNA底物(PPDS)核心将负责每个项目使用的试剂的生产，并建立产生这些试剂的程序。通过简化关键试剂的生产，如蛋白质因子、抗体和DNA底物，每个项目PI将获得额外的时间 每个项目的实验和结果都将是相似和可靠的。具体地说，PPDS核心将实现三个与服务相关的目标。 1.制备用于体内重组研究的重组DNA构建体，以及用于生产用于生化研究和抗体产生的重组蛋白。 2.哺乳动物提取物中天然蛋白的分离和特定蛋白靶标的纯化。 3.部位和病变特异性DNA底物的生产和质量控制。

Complex DNA lesions such DNA interstrand cross-links and triplehelix structures are functionally disruptive to the mammalian genome and pose unique challenges to the DNA repair system. It is most likely that repair of complex lesions involves cooperative actions of different DNA repair pathways such as homologous recombinational repair, excision repair, and mismatch repair. Our proposed program project will conduct comprehensive investigations on the processing of complex DNA lesions via molecular biology, genetics, and biochemical approaches. The Protein Factor and DNA Substrate (PPDS) Core will be responsible for the production of reagents used by each project and to establish procedures for generating these reagents. By streamlining the production of critical reagents such as protein factors, antibody, and DNA substrates, each project PI will gain additional time on experimentation and the results yielded from each project will be analogous and reliable. Specifically, the PPDS core will accomplish three service-related aims. 1. Preparation of recombinant DNA constructs for in vivo recombination studies, and for the purpose of producing recombinant proteins for biochemical studies and the generation of antibodies. 2. Fractionation of native proteins from mammalian extracts and purification of specific protein targets. 3. Production and quality control of site- and lesion-specific DNA substrates.