|Abstract: ||中文摘要 紫外線由大氣層穿越水體表層後已知會對生存於表層水體中之生物造成傷害,而紫外線於分子程級所造成之傷害主要為相鄰雙嘧啶間之共價鍵結而導致引起 DNA 扭曲變形,此種變形可使轉錄停止或使 DNA 複製後產生突變, DNA 鹼基錯誤配對亦對双螺旋結構產生局部扭曲作用。生物體為避免這種 DNA 傷害, 故有核酸切割修補和錯誤配對修補等途徑來修補受損DNA, 而辨識損害 DNA 為修復之起始步驟,因此本論文擬以小球藻和斑馬魚為水生生物模式系統,來探討水生生物中辨識扭曲DNA 螺旋結構之蛋白,並將此類辨識蛋白進行分離與鑑定。 凝膠阻抗試驗(EMSA)顯示小球藻蛋白抽取液中之 DNA錯誤配對辨識蛋白對GT配對之結合能力較其他型式之錯誤配對為高。利用二維蛋白電泳配合螢光染色發現固定化之GT 配對可吸附兩個分子量為13kDa而等電點各為5.3及5.5之 GT 辨識分子,此兩13kDa蛋白經質譜胜肽分析 (PMF)，比對Mascot資料庫並無類似蛋白存在。而等電點為5.5之蛋白經質譜蛋白定序獲得一LPSAR 短鏈胺基酸序列,發現與人類及老鼠mismatch-specific uracil/thymine-DNA glycosylase 之C 端活性區域序列有高度相似性,顯示此一蛋白質可能為小球藻之DNA glycosylase。另外小球藻蛋白抽取液內也含有紫外光傷害 DNA辨識蛋白可結合6-4光產物和環丁烷嘧啶雙體,而以二維電泳配合親和性吸附實驗,測得小球藻抽取液中存在三個分子量介於60至62 kDa間而等電點介於 5.1至5.4間之辨識蛋白,影像定量分析顯示此三蛋白對紫外線照射過之 DNA有較未照射DNA高 2 至 5倍之親和力,但取一60 kDa之辨識蛋白藉質譜分析與Mascot 資料庫比對並無發現任何類似物存在。雖然小球藻中辨識 DNA 錯誤配對與紫外光傷害之蛋白可由親和性吸附與二維蛋白電泳加以分離,但質譜分析對於蛋白身分鑑定僅能提供少許資料。 在凝膠阻抗實驗中,發現斑馬魚早期胚胎和幼魚時期之蛋白質萃取液與紫外線照射過之DNA 作用產生不同形式之蛋白質-DNA複合體。另外再經由親和性吸附與西方點墨法分析, 發現受精84 小時後之幼魚萃取液中含有類似人類XPA 之蛋白, 但在12小時胚胎之萃取液中並未發現上述蛋白質,顯示斑馬魚於早期胚胎發育時期,應未產生和已知之核酸切割修補系統(NER)類似之相關蛋白,因此斑馬魚早期胚胎中必另有其他種類之DNA傷害辨識蛋白。當從凝膠阻抗實驗的 膠片上直接將蛋白質-DNA 複合體之一小點挖下, 經膠體中trypsin消化和質譜分析,因比對分數超過有意義值,得知辨識蛋白中含有類似斑馬魚卵黃前質1 (vitellogenin I, zfVg 1) 蛋白質。於凝膠阻抗實驗之反應物中加入抗鯉魚Vg 血清,也發現能抑制12 小時斑馬魚胚胎萃取液對紫外線傷害DNA 之辨識能力, 但抑制效果僅達 40%。 為了更進一步確認辨識蛋白之特性,本論文採用製備級等電點系統 (preparative isoelectrofocusing)進行蛋白分離,結果發現此法能有效的分開具特異性與非專一性之辨識蛋白，在 pI 7-9 範圍的蛋白質對紫外線傷害DNA 有極強的辨識能力,其中以 pI 7-8 之蛋白質活性最強, 而 pI位於 4-6間之非專一性辨識蛋白則對有無受 UV照射之 DNA均具結合能力。另外將不同 pI 分離液中之蛋白予以定量後,取相同蛋白量進行分析級 2-D 電泳 (2-D electrophoresis),電泳片經由2-D 分析軟體進行影像分析後, 發現辨識活性最強之分離液中, 含有數個pI 7.3 到 7.8 而分子量為 25kDa的蛋白質點, 是其他辨識活性較弱或極弱之分離液中所沒有的,故推測這些蛋白質應為具特異性之紫外線傷害DNA 辨識蛋白。將此4個蛋白質點, 經膠體中trypsin消化和MS/MS質譜蛋白定序後發現,皆含有以下2段胺基酸序列: LPIIVTTYAK 和 IPEITMSK, 這 2 段胺基酸序列亦出現在斑馬魚卵黃前質 1-蛋白質(zfVg 1) 的 C端Lv2 區域內，生物資訊分析也發現有一由322胺基酸(GenBank,AAH94995)組成之蛋白質，其僅含有 Vg1之 Lv2 序列並且功能未知之斑馬魚蛋白，此蛋白與上述四個可能具DNA 辨識作用之蛋白，在胺基酸序列上極為相似。利用受6-4 PP扭曲之 DNA 為探針之親和性吸附實驗,證實一個 25 kDa 蛋白質點 (pI 7.4)對受傷 DNA 之辨識能力較正常 DNA 高 。西方轉印法分析發現斑馬魚早期胚胎至幼魚時期 (12-96 hr)之蛋白質萃取液含有 100 至 25 kDa 的可被抗斑馬魚Vg1 抗體辨識之蛋白，且低分子量之 Vg1類似蛋白隨斑馬魚之發育而逐漸增加，但是上述4個25 kDa 蛋白質卻對抗斑馬魚Vg1 抗體毫無反應,顯示這些低分子量類似Vg1 之蛋白應非高分子量 Vg1 之分解產物,此4 個25 kDa 蛋白質經特殊染色發現其為磷酸化蛋白 (phosphoprotein)但並非醣蛋白 (glycoprotein) 。目前由文獻已知原始的班馬魚 Vg1 分子量150 kDa,屬於 metalloprotein, 生理功能為胚胎發育過程中的營養來源,因為 Vg1 會經由蛋白酶水解,產生胺基酸和必須金屬元素以提供胚胎成長之所需。然而經由本論文發現一個與 Vg1-類似之低分子量蛋白質,其部分胺基酸序列與 Vg1 之 Lv2 具高度相似性,但此蛋白質卻展現紫外線傷害DNA 辨識作用,此蛋白之胺基酸序列顯示其極有可能為上述含322胺基酸但功能未知之斑馬魚蛋白，斑馬魚早期胚胎發育過程中產生複雜形式的 Vg 類似蛋白,尤其是某些低分子量者因其辨識扭曲 DNA 之功能,可能參與胚胎時期之核酸修補作用。|
Abstract Ultraviolet (UV) light is a component of the solar spectrum and UV penetrating from atmosphere into water is known to damage aquatic organisms present in the water surface layer. At the molecular level, UV irradiation significantly distorts the helical structure of DNA due to the formation of dipyrimidine photoproducts between two adjacent pyrimidines. To avoid producing error-prone replication or transcriptional blockage, UV-damaged DNA in most organisms is repaired primarily by excision-dependent pathways and DNA mismatches are corrected by mismatch repair. DNA repair pathways involving multipolypeptides are generally initiated by the binding of DNA damaged-recognition proteins to lesion sites. The unicellular alga Chlorella pyrenoidosa and zebrafish (Danio rerio) at fast-growing or early developmental stages are selected in this dissertation as the model systems for aquatic organisms to study proteins binding preferentially to DNA mispairs or dipyrimidine photoproducts. A duplex probe carrying a single G-T mispair was revealed by EMSA as a better binding target than those carrying other types of mispair for DNA mismatch recognition proteins in C. pyrenoidosa extracts. Two 13-kDa G-T binding polypeptides possessing pIs of 5.3 and 5.5 were isolated after resolving affinity-captured proteins by 2-D gel electrophoresis and the two factors were found to bind 5.5- and 2.8-fold stronger to heteroduplex than to homoduplex DNA, respectively. No proteins significantly homologous to the two algal G-T binding proteins were found by peptide mass fingerprinting (PMF). The sequence of a peptide generated from trypsin-cleavage of one G-T binding factor (pI 5.5) could be aligned with the amino acid sequences that form the C-terminal active sites of human and mouse mismatch-specific uracil/thymine-DNA glycosylases, suggesting the possibility of this factor as an algae- or a Chlorella-specific DNA mismatch glycosylase. UV-damaged-DNA binding activities in C. pyrenoidosa extracts recognizing both CPDs and 6-4PPs were also detected by EMSA. A 70-kDa polypeptide binding preferentially to a duplex probe carrying CPDs and 6-4PPs was pulled down from the algal extracts by affinity adsorption. Apparently higher levels of three 60 to 62-kDa polypeptides were eluted by 0.12% SDS from UV-irradiated than from unirradiated DNA immobilized on agarose beads when affinity-captured proteins were analyzed by 2-D gel electrophoresis. The 70-kDa polypeptide observed by SDS-PAGE might be an accumulation of these 60 to 62-kDa binding factors. Each SDS-eluted UV-binding polypeptide was found to bind 2 to 5-fold stronger to irradiated than to unirradiated DNA. None of the algal binding factors displayed significant sequence homology with proteins contained in the Mascot data base. Although algal proteins having higher affinities for mismatched or UV-damaged DNA were successfully isolated from C. pyrenoidosa extracts, little progress in protein identification was achieved because of the extremely low homology between algal DNA damage-recognition proteins and proteins already identified. The extracts of zebrafish (Danio rerio) early embryos and early larvae were found to contain different DNA damage-recognition proteins. Immunoblot analysis of proteins captured by agarose beads carrying a UV-irradiated duplex oligonucleotide revealed the presence of XPA in the extracts of 84-hr-old larvae, while no proteins homologous to those associated with human NER could be found in the extracts of 12-hr-old embryos. Incubation of the extracts of zebrafish embryos with a probe containing a 6-4PP produced high-shifting gel retardation complexes and participation of zebrafish vitellogenin 1(Vg1)-like proteins in generating the UV-binding activity was revealed by mass spectrometric analysis of proteins contained in the binding complexes. The binding of 12-hr old zebrafish extracts to 6-4PPs was inhibited to 60% of control by the addition of a rabbit anti-carp Vg antiserum to gel shift assay mixtures. UV-damaged-DNA binding proteins synthesized in zebrafish embryos could be efficiently separated from nonspecific DNA binding proteins by a preparative isoelectrofocusing. Nonspecific binding proteins were collected at pH from 4 to 6, yet the majority of zebrafish proteins binding preferentially to 6-4PPs were found to possess pIs about 7 to 9 and proteins displaying the strongest UV-dependent binding were isoelectrofocused between pH from 7 to 8. Four 25-kDa polypeptides having pIs between 7.3 to 7.8 were thought to be important damage-recognition factors after comparing 2-D polypeptide patterns produced by protein fractions showing strong, moderate, low and no UV-specific binding activity. Although the four polypeptides were not binding targets for a monoclonal anti-zebrafish Vg1 antibody, they all contained amino acid sequences LPIIVTTYAK and IPEITMSK which also appear in the C-terminal Lv2 region of zebrafish Vg1 according to tandem mass spectrometry(MS/MS). One 25-kDa polypeptide possessing a pI about 7.4 was shown to bind strongly to UV-damaged DNA after affinity adsorption of the active protein fraction with a 6-4PP probe and some Lv2 sequences present in other 25-kDa polypeptides were missing in this UV-binding factor. Since several 100 to 25 kDa Vtg1-like proteins produced in 12 to 96-hr-old zebrafish could be detected by the anti-zebrafish Vg1 antibody, the four 25-kDa polypeptides totally unreactive to the same antibody suggested that they originated from a source unrelated to proteolytic degradation of Vg1. The four 25-kDa polypeptides were also identified as phosphoproteins, but not glycoproteins, according to selective protein staining. The original zebrafish Vg1 having a molecular mass of 150 kDa is a metalloprotein whose degradation during developmental process is known to provide amino acids and essential metals for growing embryos. Our results revealed the identification of a group of novel embryonic proteins highly homologous to the Lv2 region of Vg1 and one low-molecular-weight Vg1-like protein might participate in DNA repair or recombination based on its preferential binding to UV-damaged DNA.