Study of human DNA (cytosine-5)-methyltransferase function in vitro

• Main Authors: Hsu, Duen-Wei, 許惇偉
• Other Authors: Shen, Che-Kun
• Format: Others
• Language: en_US
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/32709092057075831243

碩士 === 國立陽明大學 === 遺傳學研究所 === 85 === DNA甲基轉移酵素(DNA(cytosine-5)-methyltransferase or DNA MTase)廣泛存於脊椎動物，此酵素可將甲基轉至特定區域CpG位置的胞喀咬(cytosine)上，藉以調控胚胎發育、基因活性等重要功能。該酵素主要由碳端約500胺基酸長度的酵素活性區位(domain)和被認為具活性調節功能的胺端調節區位構成。 本實驗利用聚合酵素連鎖反應(PCR)技術自人類細胞K-567分離完整(full length)及胺端缺少兩個表現子(exons)的偽完整(pseudo full length)甲基轉移酵素基因，分別命名為FMT(full length MTase，4.86kb)與MT(4.5kb)；而碳端酵素活性區位已證實具新生甲基化(denovo methylation)作用，為了更了解此區位的功能，3'MT-1(2.3kb)與3'MT-2(1.5kb)兩不同長度的碳端區位被分離出。雖3'MT-1胺端有小部份由調節區位而來，但該片段蛋白卻能型新生甲基化作用。 而後再取組織蛋白(histone H1)、轉錄因子Sp 1和第一型拓撲異構酵素(topoisomnerase I)的DNA結合區位(binding domnain)接於3'MT-1的胺端以取代原有的調節區位，構築HM(Histone 3'MT-1)、SM(Sp1-3'MT-1)、TM(Topo-3'Mt-1)三種混合(hybrid)蛋白質。按著嘗試利用pKK2223-3、pET與pGEX等不同的原核生物蛋白質誘導系統大量表現這七種構築蛋白，研究它們6活體外(in vitro)的酵素活性，冀望找到適當的表現系統，以觀察FMT、MT、3'MT-1與3'MT-2甲基轉移的活性；並藉以分析具不同DNA結合區位的混合蛋白質(HM、SM與TM)能否只在特定DNA序列上行甲基轉移功能。 研究發現，SM可生成於pKK223-3系統裡，並可能有新生(de novo)甲基化作用；HM、SM與TM會表現在pET系統，但都沒有酵素活性；3'MT-1、3'MT-2、HM和SM皆可在pGEX系統中合成，唯3'MT-1才具酵素活性，且傾向新生甲基化功能。在每種表現狀態下，各蛋白質都有被嚴重分解的現象，並難以被純化，不易再做更進一步的酵素功能分析。故推測原核生物蛋白質表現系統不適於進行此研究。 DNA (cytosine-5)-methyltransferase, a DNA modifying enzyme,generally expressed in vertebrates, can transfer a methyl group to cytosine in CpG site of specific region to regulate some important function like embryogenesis, gene activity etc. This enzyme is composed an enzymatic domain at the C-terminal and a regulatory domain at the N- terminal. PCR was used to amplify the total full length (including the newly identified two exons) and pseudo full length (lacking the two exons at the N-terminal) and were named as FMT (4.86 kb) and MT (4.5 kb) respectively. The C-terminal enzymatic domain was reported to have de novo methylation function. To understand this, two C-terminal fragments were cloned and named them as 3'MT-1 (2.3 kb) and 3'MT-2 (1.5 kb). The 3'MT-1 has some regions from the N-terminal regulatory domain and for some unknown reasons this construct failed to perform methylase function. However, 3'MT-1 have the enzymatic function and favor de novo methylation. The primary aim of this thesis is to study the enzymatic function upon replacing the regulatory N-terminal region with some of the known DNA binding domains of the DNA binding proteins. For this reason the DNA binding domains of Histone H1, transcription factor Sp 1 and topoisomerase I were isolated and fused at the N-terminal region of 3'MT-1. Three hybrid constructs HM (Histone-3'MT-1), SM (Sp 1- 3'MT-1) and TM (Topo-3'MT-1) were made. Three different expression systems were used to express these hybrid constructs. After several experimental manipulations, it was found that SM can be expressed in pKK223-3 system, and had de novo methylation activity. HM, SM and TM can be expressed using pET system but all the expressed proteins lost their enzymatic activity. Finally using pGEX system, 3'MT-1, 3'MT-2, HM and SM can be syntliesized, but only 3'MT-1 had the de novo enzymatic activity. As a common feature all the constructs expressed in any of the expression system have undergone serious degradation which hampered further purification or characterization of the enzyme.