吳世雄臺灣大學：生化科學研究所李懷正Lee, Huai-ChengHuai-ChengLee2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52810具有DNA結合活性之蛋白質，通常在生物體細胞內扮演著重要角色。這類蛋白質通常直接或間接地涉及各式各樣基因表現的調控藉以維持細胞在生理上的恆定。其中具有DNA結合能力之蛋白酶即是利用降解某些轉譯因子(transcription factor)來達到正向或反向的基因調控。實驗室先前研究中，已從台灣烏來溫泉菌－嗜熱短桿菌(Brevibacillus thermoruber WR-249)中選殖出其中一種寡聚體蛋白酶－Lon蛋白酶的基因(Bt-lon)，利用遺傳工程得到大量蛋白質產物，並已完成多項酵素活性以及DNA結合活性的鑑定。本論文自其DNA結合之活性著手，探討其結合DNA序列之特異性。 Bt-lon可轉譯出分子量約為88 kDa的Lon蛋白酶(Bt-Lon)。Bt-Lon大致可分成三個功能區(domain)：N端功能區、中間ATP水解酶功能區以及C端蛋白酶功能區，其中ATP水解酶功能區還包含了受質偵測識別功\\\\\\\能區(sensor- and substrate- discrimination domain, SSD domain)。Bt-Lon 是一個具有多功能的蛋白酶，包括蛋白質的水解、ATP的水解、chaperone-like功能以及與DNA結合之能力。而Bt-Lon對受質的專一性、以及結合之DNA序列皆與大腸桿菌Lon不盡相同。值得一提的是，以往的研究中發現，大腸桿菌Lon傾向結合具有較高TG組成(TG-rich)之DNA序列，本論文實驗結果證實，Bt-Lon所結合之DNA序列之TG組成又較大腸桿菌Lon為更高。 本實驗室之前研究成果顯示，Bt-Lon的SSD功能區與DNA結合有關，為了進一步了解四級結構與DNA結合能力之間的關係，我們設計了一個Bt-Lon的刪去突變株蛋白質(truncated mutants)，然後比較這個突變株及Bt-Lon與DNA結合之能力。發現可形成四級結構之Bt-Lon較單體(monomer)之突變株有較強之DNA結合能力，這說明了在細胞內，DNA對於Lon之四級結構的形成，可能扮演著很重要的角色。DNA在被蛋白質結合後，經由DNA水解酵素(DNase I)切除未被蛋白質結合之部分，對純化出來之結合片段同時利用質譜分析儀(mass spectrometry)與選殖組件(cloning kit)進行定序(sequencing)。結果顯示，Bt-Lon與其突變株結合DNA之序列，與大腸桿菌及人類Lon所結合之序列具有相當高的一致性。近一步利用合成之寡核甘酸(oligonucleotide)與聚合酵素連鎖反應(PCR)產生之長片段DNA來確定Bt-Lon結合之DNA序列。結果顯示Bt-Lon對合成之寡核甘酸之結合能力遠低於大腸桿菌Lon，對於被結合之DNA序列也不如大腸桿菌Lon來得專一。然而，Bt-Lon對具有包含結合序列但長度大於200bp的片段，結合能力相較於其他相同長度之片段為高。綜合以上結果，Bt-Lon在選擇結合之DNA時，首先必須具有高TG含量(TG-rich)，而當DNA具有超螺旋(supercoiled)結構時則可以提升結合的強度。Proteins with DNA-binding activity mostly play an important role in the gene expression of a cell. This kind of proteins appears to exert its regulatory function by degrading some transcription factors after it binds to DNA. To investigate what kinds of elements bound by the DNA-binding proteins, identification of the binding site of the DNA must be achieved. A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized with an N-terminal domain, a central ATPase domain including a SSD (sensor- and substrate- discrimination) domain, and a C-terminal protease domain. Bt-Lon is a multi-functional enzyme and its functions include the degradation of proteins, ATPase, chaperone-like activities, and DNA binding. Gel mobility shift assays revealed that Bt-Lon binds to DNA cooperatively. In line with the previous result, we demonstrated that SSD domain is involved in the DNA-binding activity by recruiting a truncated mutant, Bt-Lon-C289. It was also found that Bt-Lon, as well as the E.coli and human Lon, exhibits a high affinity to the TG-rich DNA. To further investigate whether Bt-Lon binds DNA specifically, DNA fragments bound by Bt-Lon were purified and then subjected to MS analysis and sequence determination by cloning/sequencing on the vector (pCR4Blunt-TOPO). Experimental data revealed that the five fragments bound by Bt-Lon from plasmid pET28a were homologous in sequence with the elements bound by E. coli and human Lon. Furthermore, we selected and cloned the five fragments, encompassing ms1, ms2, lb1, lb2 and lb3 by PCR amplification. Based on the high affinity of these five fragments to Bt-Lon, it is suggested that Lon binds to DNA dependent on the site-specific TG-rich DNA sequences, and the secondary or tertiary structure of the plasmid DNA can enhance the DNA-binding affinity of Bt-Lon.ABSTRACT………………………………………………………………iii 中文摘要…………………………………………………………………v LIST OF ABBREVIATIONS……………………………………………vii INTRODUCTION……………………………………………………………1 MATERIALS AND METHODS Bacterial strains and plasmids……………………………………6 Expression and purification of Bt-Lon and its truncated mutants…………6 SDS-polyacrylamide gel electrophoresis.……………………………7 Miniprep of plasmid DNA…………………………………………………8 DNA manipulation and sequence analysis…………………………9 Gel mobility shift assays (GMSA)…………………………………9 Calculation of DNA binding cooperativity………………………9 Protein-bound DNA digestion by DNase I……………………………10 Purification of protein-bound DNA fragment…………………10 Mass spectrometry analysis………………………………………11 Insertion of DNA fragment into the cloning vector…………12 Sequence analysis……………………………………………………12 PCR solution contents and thermocycler program……………13 Double-stranded DNA annealing……………………………………14 Homology modeling of Bt-Lon-SSD…………………………………14 RESULTS PART I Characterization of DNA-binding activity of Bt-Lon………16 Determination of the Kd for DNA of Bt-Lon and its truncated muntant………16 DNA binds Bt-Lon cooperatively………………………………… 17 Characterization of composition of DNA for Lon binding………18 PART II Identification of Bt-Lon-binding DNA sequence………………20 Protection of DNA against the DNase I digestion……………20 DNA sequencing by mass spectrometry……………………………20 Identification DNA sequence by direct DNA sequencing…………21 PART III Specificity of DNA-binding of Bt-Lon……… …………………23 Binding of DNA fragments created by PCR method…………………23 Sequence comparison of Lon-bound DNA elements………………24 DISCUSSION DNA-binging activity involved in oligomerization of Bt-Lon…………26 Bt-Lon binds to higher TG-rich DNA elements than E. coli…………26 DNA-binding affinity of Lon were enhanced by the supercoiled DNA……………27 FIGURES…………………………………………………………………29 TABLES…………………………………………………………………84 REFERENCES……………………………………………………………87 APPENDIX………………………………………………………………91en-US蛋白結合之DNA序列DNA結合蛋白Lon蛋白酶DNA sequences bound by proteinDNA-binding proteinLon proteaseother