突變的T3/7 DNA 聚合酶的交互作用與功能的研究探討

Abstract

T7 DNA聚合酶是一種來自於T7噬菌體的DNA聚合酶，它是一種需要DNA當作模板的DNA聚合酶。由於T7複製系統是一種簡單且高效率的DNA複製系統，因此此系統模型廣泛應用於科學研究，藉此了解DNA分子、DNA聚合酶與其輔助蛋白在DNA複製系統中的角色。T7 DNA聚合酶有兩個次單元，分別是由T7噬菌體的基因5所表達的蛋白和宿主(大腸桿菌)自身原有的硫氧還蛋白所組成。根據科學家的研究，T7噬菌體與T3噬菌體的基因5蛋白有同源性，在胺基酸序列上有大約97%相同。 本論文依據T7複製系統之模型，研究T3/7重組噬菌體的基因5蛋白。T3/7重組噬菌體在定序資料上顯示，基因5 在DNA序列上大部分以T3噬菌體DNA為主，有99%以上的相似性，在胺基酸序列上只有4個不同處。 在前人的實驗成果中發現，有幾株在基因5上有突變的T3/7重組噬菌體，會對其複製造成影響。因此延續其成果，我利用大腸桿菌表現這些突變的基因5蛋白，再利用DNA親合管柱分析這些突變的蛋白對DNA的結合能力。我經由DNA親合管柱分析，比對這些突變的基因5蛋白與野生型基因5蛋白對DNA的結合能力，發現某些突變的基因5蛋白的DNA結合能力明顯不同於野生型。 我利用DNA定序與3D結構模型的比對，預測基因5蛋白的那個殘基對DNA結合能力來說是重要的。其中經由T7 DNA聚合酶的3D結構模型比對發現，胺基酸序列364~367很靠近DNA結合的位置。且根據DNA親合管柱分析的結果，胺基酸序列364~367可能是基因5蛋白跟DNA發生作用的關鍵區域。 我成功的在大腸桿菌中表現T3/7噬菌體的野生型基因5蛋白與其突變株的基因5蛋白，並純化基因5蛋白，以及分析這些突變基因5蛋白與DNA間的作用關係。希望未來後人能夠利用這些研究資料，來了解這些突變位置對DNA聚合酶的影響。

The T7 DNA polymerase of the T7 bacteriophage is a DNA-dependent DNA polymerase. Due to T7 replication system is a simple and efficient DNA replication system, so this system model is widely used in scientific research to understand about the role of DNA molecule, DNA polymerase and accessory proteins in DNA replication system. T7 DNA polymerase has two subunit – the Gene 5 protein (gp5) encoded by bacteriophage T7 and the thioredoxin (trx) produced by host (E.coli). According to the research of scientists, the gene 5 protein of bacteriophage T3 and T7 are homologues, about 97% similarities in the amino acid sequence level. In this paper, the research of Gene 5 protein of T3 / T7 recombinant phage was based on the T7 replication system model. The DNA sequencing data show that Gene 5 protein of T3 / T7 recombinant phage relies mainly on T3 phage DNA sequence, there are more than 99% similarity, and only four differences in the amino acid sequence. In the results of previous experiments have discovered that the some Gene 5 mutant strains of T3 / T7 recombinant phage that affect their own replication. Therefore, the continuation of the results. I express the mutant Gene 5 protein in the E.coli, and then using DNA-affinity chromatography to analyze the DNA binding capacity of these mutant proteins. I compared the DNA binding capacity of wild-type Gene 5 protein with mutant Gene 5 proteins by DNA-affinity chromatography and found that DNA binding capacity of some mutant Gene 5 proteins are definitely different from wild type. I employed DNA sequencing and 3D model comparison to predict which residues are important for Gene 5 protein to bind DNA. Through the T7 DNA polymerase 3D structural model comparison found that the amino acid residues at positions 364 to 367 of Gene 5 protein are very close to the bound DNA. And in accordance with the results of the DNA affinity chromatography, maybe the amino acid residues at positions 364 to 367 of Gene 5 protein are the key region for interaction with DNA. I successfully express the wild-type Gene 5 protein of T3 / T7 phage and mutant Gene 5 protein in E. coli , and purified Gene 5 protein. And I analyzed the interaction of these mutant Gene 5 proteins with DNA. I hope these research data are useful in understanding that effects of these mutant positions on the DNA polymerase in the future.