Hoogsteen base pairing in human DNA polymerase iota

描述（由申请人提供）：长期目标是描述人类DNA聚合酶i （Poll）对未受损和受损DNA的作用机制。该项目的具体目的是通过Poll研究Hoogsteen碱基配对在未损伤和受损dna复制中的作用，并且拟议的研究将使用生化和结构相结合的方法，hi Aim 1，生化研究将使用腺嘌呤和鸟嘌呤的碱基类似物来分析模板嘌呤中不同的氢键受体/供体对其与进入的嘧啶核苷酸（nt）的Hoogsteen配对的贡献。目标2将检验假设，即Hoogsteen碱基配对使Poll能够合并相反的DNA损伤，这些损伤严重影响模板碱基的小凹槽或损害其沃森-克里克碱基配对能力。Poll的熟练程度，以合并nts对面的病变，并从那里延伸将由稳态动力学方法决定。Aim 3将通过分析与Poll三元结构中模板nt或传入nt相互作用的手指和手掌结构域的突变变化对nt结合的效率和保真度的影响，确定Poll活性位点中Hoogsteen碱基配对的结构碱基；Aim 4，具有未损坏模板G， T和C的Poll三元配合物的x射线结构；在Aim 5中，将确定具有严重冲击小凹槽或影响沃森-克里克碱基配对的DNA的模板位点上的损伤的Poll的三元结构，以及存在不同的输入nts的其他结构。

DESCRIPTION (provided by applicant): The long term objectives are to delineate the action mechanisms of human DNA polymerase i (Poll) on undamaged and damaged DNAs. The specific aims of this project are to examine the role of Hoogsteen base pairing in the replication of undamaged and damaged DNAs by Poll, and the proposed studies will use a combined biochemical and structural approach, hi Aim 1, biochemical studies will be done with base analogs of adenine and guanine to analyze the contributions of different hydrogen bond acceptors/donors in template purines to their Hoogsteen pairing with the incoming pyrimidine nucleotide (nt). Aim 2 will test the hypothesis that Hoogsteen base pairing enables Poll to incorporate nts opposite DNA lesions that severely impinge upon the minor groove of the template base or which impair its Watson-Crick base pairing ability. The proficiency of Poll for incorporating nts opposite such lesions and to extend there from will be determined by steady-state kinetic methods. Aim 3 will determine the structural bases of Hoogsteen base pairing in Poll's active site by analyzing the effects that mutational changes in the residues of the fingers and palm domains, that are seen to interact with the templating nt or with the incoming nt in Poll ternary structure, have on the efficiency and fidelity of nt incorporation, hi Aim 4, the X-ray structures of ternary complexes of Poll with undamaged templates G, T, and C, in the presence of different incoming nts will be determined, and in Aim 5, the ternary structures of Poll with DNA containing lesions at the templating site that severely impinge upon the minor groove, or which affect Watson-Crick base pairing, and others, in the presence of different incoming nts will be determined. The results will be highly relevant for cancer biology, as the manner in which DNA lesions are bypassed during replication has a major impact on genome stability; and, in fact, the inactivation of Polr in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum.