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。本项目的具体目的是研究Hoogsteen碱基配对在Poll复制未受损和受损DNA中的作用，拟议的研究将使用生化和结构方法相结合的方法。在目标1中，将对腺嘌呤和鸟嘌呤的碱基类似物进行生化研究，以分析模板嘌呤中不同的氢键受体/供体对其Hoogsteen与输入的嘧啶核苷酸(NT)配对的贡献。目标2将测试假设，即Hoogsteen碱基配对使Poll能够整合NTS相反的DNA损伤，这些损伤严重冲击模板碱基的小沟或损害其Watson-Crick碱基配对能力。Poll的熟练程度将通过稳态动力学方法确定，以将NTS与这些病变相对并从中延伸。目的3将通过分析手指残基和手掌结构域的突变变化对NT掺入的效率和保真度的影响来确定Poll活性部位的Hoogsteen碱基配对的结构基础。在目标4中，将确定Poll与未损坏的模板G、T和C在不同传入NTS存在下的三元络合物的X射线结构，在目标5中，Poll的三元结构与严重冲击小沟槽的模板位置处的含有DNA的损伤相结合，或者哪些影响Watson-Crick碱基配对，以及在不同传入NTS存在的情况下的其他将被确定。 这一结果将与癌症生物学高度相关，因为在复制过程中绕过DNA损伤的方式对基因组稳定性有重大影响；事实上，人类POLR的失活会导致癌症倾向综合征，这是着色性干皮病的变体。

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.