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性，并对DNA修复系统提出了独特的挑战。最有可能的是，复杂损伤的修复涉及不同DNA修复途径的协同作用，例如同源重组修复、切除修复和错配修复。我们建议的计划项目将通过分子生物学，遗传学和生物化学方法对复杂的DNA损伤的处理进行全面的调查。蛋白质因子和DNA底物（PPDS）核心将负责生产每个项目所用的试剂，并建立生成这些试剂的程序。通过简化关键试剂（如蛋白因子、抗体和DNA底物）的生产，每个项目PI将获得额外的时间， 实验和每个项目产生的结果将是类似的和可靠的。具体而言，PPDS核心将实现三个与服务有关的目标。 1.用于体内重组研究的重组DNA构建体的制备，以及用于生产用于生物化学研究的重组蛋白和抗体生成的目的。 2.从哺乳动物提取物中分离天然蛋白质和纯化特异性蛋白质靶标。 3.位点特异性和病变特异性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.