In Vitro and In Vivo processing of 8-Oxoguanine and Abasic Sites Introduced Into Putative Mutagenic Hotspots by DNA Polymerases and Base Excision Repair Enzymes

Hatahet序列上下文在DNA自发突变和损伤诱导突变的产生中起重要作用，并在体内通过诱变热点和冷点表现出来。自由基损伤的大量产物是8-氧鸟嘌呤（8-oxoG），与诱变密切相关。这种病变已被证明在体外与A错配，并在体内引起G(r)T转化。8-oxoG通过碱基切除修复途径进行修复。为了在体外分离假定的致突变热点，病变被引入寡核苷酸，每侧四个邻居被完全随机化。根据其增加错误编码频率和/或降低损伤修复效率的能力分离序列。其中一个这样的序列，5' G(8-oxoG)A显示出甲酰胺嘧啶DNA n -糖基化酶（Fpg）去除8-oxoG的能力降低了30倍，与其他序列背景相比，在病变的相反方向错配A的频率增加了15倍。综上所述，这两种效应预测了8-oxoG在该序列背景下诱变潜力的显著增加。这个项目有两个目标。首先，了解假设热点中的核苷酸在DNA合成过程中影响碱基切除修复酶对病变的不良识别和/或更高的错误编码的机制。为了做到这一点，8-oxoG的致突变潜力将在5‘ g （8-oxoG）之间进行比较，这是一个假定的热点，并且在5’或3'邻居被改变为以下嘌呤类似物之一的情况下：次黄嘌呤，nebularine， 2-氨基嘌呤或2,6-二氨基嘌呤。这些邻居对损伤修复效率的影响将有助于确定不同嘌呤外环基团与Fpg之间的具体相互作用。类似地，与DNA聚合酶的特定相互作用将通过比较8-oxoG的错误编码效率作为将最近邻居改变为这些类似物之一的函数来剖析。该项目的第二个目标是在体内验证上述体外相互作用。含有8-氧鸟嘌呤的寡核苷酸被嘌呤或嘌呤类似物包围在两侧，将被引入质粒，质粒反过来将转化为精通或缺乏8-氧鸟嘌呤修复和/或DNA聚合酶校对的大肠杆菌菌株。除了获得有关序列背景作用的信息外，在不同细胞背景下测量8-oxoG诱导的突变频率应该非常有助于评估无效损伤修复和错误DNA合成对突变的相对贡献。因此，这个项目应该对细胞维持其遗传完整性的基本机制产生有益的见解。本研究的目的是了解氧化损伤诱导大肠杆菌诱变的机制。突变是DNA的永久变化，DNA是储存所有细胞功能和繁殖所需信息的分子。因此，突变对生物体的健康是有害的。反过来，突变发生的关键因素是DNA损伤。多种因素导致DNA损伤，包括电离和紫外线辐射、化学氧化剂和正常的细胞代谢。因此，所有的生物体都进化出了修复DNA的系统，这并不奇怪，研究这样的系统将积极地有助于解决由突变引起的问题。这个项目的目标是更好地理解这个修复系统的功能，使用细菌和无细胞系统。

9728084 Hatahet Sequence context plays an important role in the generation of spontaneous and damage-induced mutations in DNA, and is manifested in vivo by mutagenic hot- and coldspots. An abundant product of free radical damage which has been strongly implicated in mutagenesis is 8-oxoguanine (8-oxoG). This lesion has been shown to mispair with A in vitro and to cause G(r)T transversions in vivo. 8-oxoG is repaired by the base excision repair pathway. To isolate putative mutagenic hotspots in vitro, the lesion has been introduced into an oligonucleotide where four neighbors on each side have been completely randomized. Sequences were isolated based on their ability to increase the miscoding frequency and/or reduce the efficiency of lesion repair. One such sequence, 5' G(8-oxoG)A shows up to 30 fold reduction in the ability of formamidopyrimidine DNA N-glycosylase (Fpg) to remove 8-oxoG and up to 15 fold increase in the frequency of misincorporating A opposite the lesion as compared to other sequence contexts. Combined, these two effects predict a significant increase in the mutagenic potential of 8-oxoG within this sequence context. This project has two objectives. First, to understand the mechanism by which the nucleotides in a putative hotspot effect poor recognition of a lesion by base excision repair enzymes and/or higher miscoding during DNA synthesis. To do that, the mutagenic potential of 8-oxoG will be compared between the 5'G(8-oxoG)A putative hotspot and contexts where either the 5' or 3' neighbor is changed to one of the following purine analogs: hypoxanthine, nebularine, 2-aminopurine, or 2,6-diaminopurine. The effect of each of these neighbors on lesion repair efficiency will help define specific interactions between the different purine exocyclic groups and Fpg. Similarly, specific interactions with DNA polymerase will be dissected by comparing the miscoding efficiency of 8-oxoG as a function of changing the nearest neighbors to one of these analogs. The second o bjective of this project is to verify the above in vitro interactions in vivo. Oligonucleotides containing 8-oxoguanine surrounded by purines or purine analogs on both sides will be introduced into plasmids which in turn will be transformed into E. coli strains proficient or deficient in repair of 8-oxoguanine and/or DNA polymerase proofreading. In addition to gaining information about the role of sequence context, measurement of 8-oxoG induced mutation frequency in the different cellular backgrounds should be very helpful in assessing the relative contribution of inefficient lesion repair and erroneous DNA synthesis to mutagenesis. As such, this project should yield helpful insights into the fundamental mechanisms by which cells maintain their genetic integrity. The goal of this research is to understand the mechanism of oxidation damage-induced mutagenesis in E. coli. Mutations are permanent changes in DNA, the molecule which stores the information needed by all cells to function and reproduce. As such, mutations can be detrimental to an organism's well-being. In turn, a key player in the development of mutations is DNA damage. A wide range of factors contribute to DNA damage including ionizing and ultraviolet radiation, chemical oxidants and normal cellular metabolism. It is therefore not surprising that all organisms have evolved systems which repair DNA, and studying such systems would positively contribute to solving problems caused by mutations. The goal of this project is to better understand the function of this repair system using bacteria and a cell-free system.