Analysis of Eucaryotic DNA Polymerase

本提案的目的是研究真核生物DNA聚合酶与其引物模板的相互作用。我们的方法包括将寡核苷酸引物模板与多亚基DNA聚合酶的活性位点肽进行光交联。DNA聚合酶α引物酶活性和DNA聚合酶epsilon的3‘ - 5’外切酶活性位点将使用这一基本程序的修改进行研究。两种不同类型的光活性核苷酸类似物将在这项工作中使用。在第一种方法中，（5-叠氮- dUTP， 8-叠氮- datp和8-叠氮- dgtp）光活性叠氮基团直接连接到嘧啶或嘌呤碱上。这个去保护的寡核苷酸的初级氨基随后将与一系列光活性、异双功能交联试剂反应，生成具有特定位置、可切割、光活性核苷酸类似物的寡核苷酸引物。本研究的最终目的是通过序列分析分离和鉴定特异性活性位点的交联寡核苷酸引物模板。通过首先将DNA聚合酶的活性位点映射到复合物的特定亚基，然后映射到亚基内的特定色氨酸，我们希望开始了解这种非凡的酶复合物的结构和功能组织。

The goal of this proposal is to investigate the interaction of a eucaryotic DNA polymerase with its primer-template. Our approach involves photo-crosslinking of oligonucleotide primer- templates to active site peptides of the multi-subunit DNA polymerase. The DNA primase activity of polymerase alpha and the 3' - 5' exonuclease active site of DNA polymerase epsilon will be investigated using modifications of this basic procedure. Two different classes of photoactive nucleotide analogs will be used in this work. In the first, (5-azido- dUTP, 8-azido-dATP and 8-azido-dGTP) the photoactive azido group is attached directly to the pyrimidine or purine base. The primary amino group of this deprotected oligonucleotide will then be reacted with a series of photoactive, heterobifunctional crosslinking reagents to generate oligonucleotide primers with specifically-positioned, cleavable, photoactive nucleotide analogs. The ultimate goal of this proposal is to isolate and identify by sequence analysis, specific active site tryptic peptides crosslinked to oligonucleotide primer-templates. By mapping the active sites of DNA polymerase first to specific subunits of the complex, and later to specific tryptic peptides within the subunit, we hope to begin to understand the structural and functional organization of this remarkable enzyme complex.