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

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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.