Abstract
Ku 自體免疫抗原（autoantigen）是由 Ku70 及 Ku80 兩個次單體所組成的一個蛋白質複合體。目前已知 Ku 蛋白質參與去氧核糖核酸雙股斷裂的修復（DNA double-strand break repair）以及 V(D)J 重組《V(D)Jrecombination》的過程。同時， Ku protein 是去氧核糖核酸依存性磷酸化酵素（DNA dependent protein kinase，簡稱 DNA-PK）中負責與去氧核糖核酸端點結合（DNA end-binding, DEB）的構成要素之一，而DNA-PK 的另一個負責磷酸化反應的則是 460 kDa 的 DNA-Pkcs(catalytic subunit)。現在的證據更顯示 DNA-PK 可以在體外（invitro）磷酸化許多參與複製（replication），轉錄（transcription）和修補（repair）作用的核蛋白質。 為了研究 Ku protein 在去氧核糖核酸修復作用中的生化機制，本實驗室需要表現大量的 Ku70 和 Ku80protein 。首先利用反轉錄酵素及聚合酵素連鎖反應的方法（reversetranscription- polymerase chain reaction, RT-PCR）來合成具有完整遺傳字碼區域（coding region）的人類 Ku70 互補 DNA （hKu70 cDNA）。接下來便將這段 cDNA 嵌入一個在細菌中表現的載體（ vector ），pQE30 。 pQE30 上具有一乳糖誘導啟動子（galactose- induciblepromoter），因此，以 IPTG (isopropyl-beta-D-thiogalactopyranoside)誘導後，便可以在此細菌系統中得到大量在 N端帶有六個組氨酸（histidine）的人類 Ku70 融合蛋白質（fusionprotein）。利用以上述的方法，吾人成功地在細菌中表現了大量 Ku70蛋白質。DNA部份定序及西方氏墨點轉漬法的結果都證明所表現的確實是Ku70。 此外，本論文也研究不同細胞株（cell lines）中 Kuprotein 的 DNA 端點結合（DEB）性質。Ku 的 DEB 活性是分別使用抽取自老鼠細胞株 NIH/3T3，中國倉鼠卵巢細胞 CHO K1，人類子宮頸癌細胞HeLa 和人類結腸腺癌細胞 RKO 的粗萃取物（crude extract），以電泳泳動偏移測定（electrophoretic mobility shift assay, EMSA）加以偵測。實驗結果顯示：當使用長度為 222 bp 的DNA 探針（probe）時，不同 cell lines 中所形成的 Ku-DNA 複合體，在電泳膠片上呈現不同程度的偏移。而偏移的程度依序是： NIH/3T3 （最慢）， CHO K1 （次之），而 HeLa 和 RKO （最快）的偏移位置則相同。然而，當使用長度為 30bp 的DNA 探針時，不同 cell lines 中的 Ku-DNA complexes 則在膠片上偏移到相同高度的位置。更進一步的實驗證據顯示，當所使用的 probe長度大於 60 bp 時，便可觀察到不同程度的偏移現象，同時，這個有趣的現象並非是由於某段特別的 DNA 序列（ sequence ）所造成的。一些實驗數據指出不同 cell lines 中的 Ku 蛋白質的分子量是相當的，因此，推論在不同 cell lines 中存在著某個大小互異的蛋白質，該蛋白質會與 Ku 和一小段 DNA 區域形成 complex ，而造成不同 cell lines 的DEB complexes 偏移位置不同的結果。萃取物混合試驗的結果支持這樣的假設，但最終的證明仍需要更進一步的實驗來驗證。 本實驗亦對人類二倍體纖維原細胞株（human diploid fibroblast cell lines） HF ，AG1521 和腫瘤細胞株 HeLa ， RKO 的 Ku DEB 性質加以比較。結果說明HF 和 AG1521 具有兩種 DEB 活性，其中一個所形成的 DNA-proteincomplex 以 EMSA 測定所偏移的位置和 HeLa 及 RKO 的相同，另一個則有較快的偏移程度。紫外光交叉結合（UV cross-linking）和西方氏墨點轉漬法（Western blot）的結果說明形成偏移較快複合體之蛋白質為Ku70 和分子量為 69 kDa 次單體（subunit）所組成之雜二聚體，吾人命名為 KuJ。KuJ 與 Ku 蛋白質的 DNA 結合特性非常相似，但磷酸化及去磷酸化作用對這兩個蛋白質生合成（biosynthesis）的影響卻是不同，此點暗示，調控 KuJ 與 Ku 蛋白質表現、生成的機制是不一樣的，而 KuJ可能扮演著與 Ku 不同的特殊角色。 本論文研究不同細胞株中所表現的雙股 DNA 端點結合活性，目的在探索 Ku 蛋白質於生物體中所扮演之角色，及其作用時的可能途徑、機制，而吾人之實驗結果可提供一些方向。The Ku autoimmune antigen is a complex composed of twosubunits of Ku70 and Ku80. Ku protein is involved in DNAdouble-strand break repair andV(D)J recombination and is the DNAend-binding (DEB) component of the DNA-dependent protein kinase（DNA-PK）. The 460 kDa catalytic subunit, DNA-PKcs, of DNA-PKcan phosphorylate many nuclear proteins involved in replication,transcription and repair in vitro. In order to study thebiochemical mechanism of Ku in DNA repair, we wouldlike toexpress both Ku70 and Ku80 proteins in large quantity. ThehumanKu70 cDNA containing the whole coding region was generatedby reverse transcription coupled polymerase chain reaction (RT-PCR). The hKu70 cDNA was further subcloned into a bacterialexpression vector, pQE30. After IPTG treatment, we couldsuccessfully express large quantity of human Ku70 protein inthis system. In addition, we also investigated the DEBproperty of Ku protein from different cell lines. The DEBactivity of Ku from NIH/3T3, CHO K1, HeLa and RKO cells wascharacterized by using the crude extract and electrophoreticmobility shift assay. Ku proteins from those cell lines causeddifferent mobility shift of the Ku-DNA complex when the DNAprobe was 222 bp long. Thus, the degree of retardation was inthe order of NIH/3T3 (the highest), CHO K1, and the two humancell line HeLa and RKO which had the same degree of mobilityshift. However, the complexes generated by those Ku proteinswere shifted to the same position when the DNA probe was 30 bplong. Further studies showed that the different degree ofretardation could be seen when the probe was 60 bp or longer andthe difference was independent of DNA sequence. Severalexperiments were carried out to show that the size of thosedifferent Ku proteins is the same. It seems that anotherprotein, with different size in different cells, causes theobserved different retardation. This protein requires Ku and ashort DNA region to exert its effect. The results of a mixingexperiment using a fixed amount K1 Ku and increasing amount ofHeLa Ku support this hypothesis. Further experiments will beperformed to confirm this preliminary finding. The DEBproperty of Ku in human diploid fibroblast cell lines HF,AG1521, and tumor cell lines HeLa and RKO was studied. Theresults indicated that HF and AG1521 have two DEB activities.Thus, one of the DNA-protein complex migrated to the sameposition as the HeLa and RKO complex while the other complex hada faster migration. The faster-migrating complex could besupper-shifted by anti-Ku70 and anti-Ku80 antibodies. UV cross-linking experiment indicated the DNA-binding component was the70 kDa subunit as in the case of HeLa cells. Furthermore,Western analysis showed that besides two immune-reactiveproteins of 70 and 80 kDa, there was another protein of 69 kDain HF cells. Taken together, our results suggest that in humandiploid fibroblast, there is another protein having the same DEBactivity as Ku. This protein (KuJ) is Ku- related and is aheterodimer composed of two subunits of 70 kDa (Ku70) and 69 kDa(Ku60). Many DEB characteristics of KuJ and Ku are the same,indicating DNA binding properties of those two proteins are verysimilar. However, the amount of KuJ and therefore DEB activitywere found regulated by phosphorylation/dephosphorylation whileKu was not. This fact combined with the finding that KuJ isexpressed in human diploid cell lines only suggests that KuJmight have another function which is uniquely required in thisparticular cell type.In conclusion, we have determined the DNA-end-binding properties of Ku from different cell lines. Thedata presented here may provide clues to the biologicalfunctions and mechanism of these proteins in DNA repair.