Protein Purification and DNA Substrates Core

复杂的DNA损伤，如DNA链间交联和三螺旋结构，对哺乳动物基因组具有功能破坏作用，对DNA修复系统提出了独特的挑战。复杂病变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。本项目拟通过分子生物学、遗传学和生物化学等方法对复杂DNA病变的处理进行综合研究。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，并建立生成这些试剂的程序。通过简化关键试剂如蛋白质因子、抗体和DNA底物的生产，每个项目PI将获得额外的时间

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.