BYPASS FIDELITY OF OXIDATIVE DAMAGE LESIONS BY Y-FAMILY DNA POLYMERASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 基因组的完整性关键取决于DNA复制的保真度和效率。复制基因组DNA的高保真DNA聚合酶可以在某些DNA损伤部位停滞不前，一个或多个病变旁路Y家族聚合酶被招募来转移病变。这种旁路聚合酶对未损伤的DNA模板具有较高的错误率和较低的处理能力，但可以在病变部位对面插入碱基，并以无错误(避免突变)或易出错(产生突变)的方式从损坏的碱基对延伸。 我们的目标是了解定义与旁路聚合酶复制氧化损伤损伤相关的突变事件的分子机制。患癌症的风险增加与氧化应激有关，氧化应激是由于细胞对炎症和感染的反应导致活性氧产生过多。基因组DNA中氧化损伤水平的升高也与神经退行性疾病、衰老和心血管疾病有关。 我们的结晶学研究是在最普遍的氧化损伤损伤上进行的，即8-氧鸟嘌呤，稳定的开环5-胍-4-硝基咪唑加合物和融合的双环螺亚胺二乙内酰脲加合物，放置在Dpo4 Y-家族聚合酶活性位点的DNA模板链的背景下。 此前，我们的团队发现了一些结构和功能特征，这些特征使低保真的Dpo4聚合酶能够实现与8-氧鸟嘌呤(OxoG)损伤相反的胞嘧啶碱基的无错误插入。我们发现，oxoG损伤的Dpo4活性部位采用了与dCTP配对所必需的反构象，识别事件由Dpo4氨基酸残基与oxoG的多次有利接触促进。我们还证明了DPO4在整个催化循环中经历了不同于在高保真聚合酶中观察到的相应易位事件的逐步易位。我们的努力应该阐明促进相对于和越过不同大小和形状的氧化损伤损伤的无错误或容易错误的DNA合成的共同因素，以及损伤结构和首选构象。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Genomic integrity depends critically on the fidelity and efficiency of DNA replication. High-fidelity DNA polymerases that replicate genomic DNA can stall on certain DNA damage sites, and one or more lesion-bypass Y-family polymerases are recruited to transit the lesion. Such bypass polymerases have a higher error rate and lower processivity on undamaged DNA templates, but can insert a base opposite a lesion site and extend from a damaged base pair in error-free (mutation-avoiding) or error-prone (mutation-generating) manner. Our goal is to understand the molecular mechanisms that define the mutagenic events associated with replication of oxidative damage lesions by bypass polymerases. An increased risk for developing cancer has been linked to oxidative stress due to the overproduction of reactive oxygen species resulting from the response of cells to inflammation and infection. Elevated levels of oxidative damage lesions in genomic DNA have also been associated with neurodegenerative diseases, aging, and cardiovascular disorders. Our crystallographic studies are undertaken on the most prevalent oxidative damage lesions, namely, 8-oxoguanine, the stable ring-opened 5-guanidino-4-nitroimidazole adduct and the fused bicyclic spiroiminodihydantoin adduct, placed in the context of DNA template strands of the active site of Dpo4 Y-family polymerase. Previously, our group uncovered structural and functional features that enable low-fidelity Dpo4 polymerase to achieve predominantly error-free insertion of a cytosine base opposite the 8-oxoguanine (oxoG) lesion. We found of The oxoG lesion adopts an anti conformation within the Dpo4 active site, that is necessary for pairing with dCTP, with the recognition event facilitated by multiple and favorable contacts of Dpo4 amino acid residues with the oxoG. We have also demonstrated that Dpo4 undergoes stepwise translocations throughout the catalytic cycle that are distinct from the corresponding translocation events observed in high-fidelity polymerases. Our efforts should elucidate the common factors that promote error-free or error-prone DNA synthesis opposite and past the oxidative damage lesions of varying size and shape, as well as lesion architecture and preferred conformations.