Hoogsteen base pairing in human DNA polymerase iota

描述（由申请人提供）：长期目标是描述人DNA聚合酶i（Poll）对未受损和受损DNA的作用机制。该项目的具体目标是通过Poll检查Hoogsteen碱基配对在未受损和受损DNA复制中的作用，拟议的研究将使用生物化学和结构相结合的方法，hi Aim 1，将用腺嘌呤和鸟嘌呤的碱基类似物进行生物化学研究，以分析不同氢键受体的贡献。模板嘌呤中的供体与进入的嘧啶核苷酸（nt）进行Hoogsteen配对。目的2将检验Hoogsteen碱基配对使Poll能够掺入与模板碱基的小沟严重冲突或损害其Watson-Crick碱基配对能力的DNA损伤相对的nts的假设。将通过稳态动力学方法确定Poll合并此类病变对面的nt并从其延伸的能力。目的3将通过分析手指和手掌结构域的残基中的突变变化对nt掺入的效率和保真度的影响来确定Poll活性位点中Hoogsteen碱基配对的结构基础，所述手指和手掌结构域被认为与模板nt或与Poll三元结构中的传入nt相互作用。将测定Poll与未损坏的模板G、T和C的三元复合物在不同输入nts存在下的X射线结构，并且在目标5中，Poll的三元结构，在模板位点含有严重影响小沟或影响Watson-Crick碱基配对的损伤的DNA，以及其他，将确定是否存在不同的输入NT。 该结果与癌症生物学高度相关，因为复制过程中绕过DNA损伤的方式对基因组稳定性具有重大影响;事实上，人类Polr的失活会导致癌症易感综合征，即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.