RPA (replication protein A)為一單股DNA結合蛋白，普遍存於真核細胞之細胞核中，主要是由RPA1、RPA2及RPA3三個次體組成一穩定複合物。目前所知，RPA對於單股及受損之DNA有高度親和性，且其功能牽涉到DNA複製、修補以及基因重組等生理反應。此外，RPA2的磷酸化也扮演著重要的角色。當細胞進入S phase時，RPA2會有磷酸化的現象產生，直到M phase的晚期才去磷酸；而當細胞遭受到紫外光(UV)、游離輻射(IR)的照射，或是施以DNA複製抑制劑而對細胞造成損傷時，RPA2被觀察到有高度磷酸化現象的發生。而磷酸化態的RPA2可能會將DNA損傷的訊息傳遞並連結至S-phase check point機制，使p53從RPA-p53複合物中釋出。由以上這些現象可看出RPA2及其磷酸化現象在DNA代謝過程或是調控細胞週期上扮演著重要的角色。然而直至目前為止RPA2的生理角色仍未清楚的被了解。因此，為了探討RPA2在細胞中及其在生理上可能角色之扮演，本實驗室試著利用Yeast-two hybrid之方法，企圖找出其他會與RPA2有交互結合作用之蛋白，希望從而更能了解RPA2磷酸化與其他結合蛋白之交互作用的生理意義。篩選之結果成功地從人類乳腺細胞基因庫中篩選出二個與RPA2有較為顯著交互作用之蛋白： Tyro 3 (又稱為Sky、Rse、Tif、Dtk、Brk、Etk2) 和4E-BP3 (eIF4E-binding protein 3)。研究指出，Tyro 3是屬於Axl/Sky/Mer RTK (Receptor Tyrosin kinase)家族之一員，已知在細胞生長、分化及代謝上扮演著相當重要的調控角色。因此本實驗利用Yeast-two Hybrid以及免疫沈澱之方法，二者皆證實了RPA2及Tyro 3之間確實存在著直接交互結合作用。此結果或許意味著RPA2參與到Tyro 3所調控之細胞生長、分化和代謝之生理反應中；又或者是Tyro 3亦存在著其他的生理意義。而在另一個利用Yeast-two Hybrid方法被篩選出疑似與RPA2有交互作用之蛋白4E-BP3，已知屬於4E-BPs家族之一員，其磷酸化的型態會與eIF4G競爭eIF4E的結合位置，使得cap-mRNA無法帶入43S pre-initiation complex，進而抑制轉譯的速率，對於細胞生長有負向調節的功能。我們初步的實驗證實RPA2與4E-BP3兩者有交互作用，顯示4E-BP3亦可能在細胞中參與調控RPA2的功能。因此，本實驗利用免疫沈澱法再次證實了RPA2和4E-BP3之間的交互結合作用。而為了更進一步縮小範圍了解其間的交互作用之位置，因此將RPA2之三大特殊功能區分別截短(deletion)並構築於帶有T7啟動子(promoter)之載體上，由TnT quick coupled transcription/translation system於in vitro情形下表現出蛋白質，接著經由免疫沈澱法分析，結果發現4E-BP3會與全長以及截斷C端(170-270胺基酸)之RPA2有交互結合作用。因此推論RPA2和4E-BP3之交互結合位置應位於RPA2之N端磷酸化區和中段DNA結合區之間。此外，實驗室之前發現利用細胞受曝曬高劑量(50 J/m2)紫外光之刺激模式，會造成原本在正常情況下具有交互結合作用之RPA2、4E-BP3複合體分開。為更進一步探討可能影響其結合作用消失之因素為何？因此將經過紫外光曝曬處理過後之HEK293細胞，依不同時間點收集細胞萃取液，並進一步將細胞質和細胞核之蛋白分離，經由西方點漬法(Western Blot)分析RPA2之蛋白變化量，發現在照射後二個小時內RPA2蛋白沒有明顯的變化。綜合言之，本論文的實驗結可証明RPA2和Tyro 3、4E-BP3皆有交互作用。而RPA2和4E-BP3之結合作用在細胞受紫外光照射後即會消失，而此現象並非是RPA2蛋白被裂解(degradation)或移轉(translocation)造成的。進一步探討其結合區域，結果發現是位於RPA2胺基酸1-170之內，這中間包含了RPA2磷酸化區及DNA結合區。

RPA (replication protein A), a ubiquitous eukaryotic single-stranded DNA binding protein complex, which is composed of RPA1, RPA2 and RPA3 subunits. The heterotrimeric complex has a high affinity for single stranded and damage DNA, and plays essential roles in DNA replication, repair and recombination. Furthermore, RPA function is mediated by its phosphorylation and protein-protein interactions. RPA2 is phosphorylated in a cell cycle-dependent manner and in response to DNA damage, such as UV or ionizing irradiation, and treated with replication inhibitors. The phosphorylation of RPA2 may elicit the signaling transduction during DNA damage and links to the S-phase checkpoint mechanism. The purpose of our study is to identify the RPA2 binding proteins and elucidate the biochemical and functional characteristics of RPA2 phosphorylation. In our previous studies, two RPA2-binding proteins, Tyro 3 (also named Rse, Sky, Brt, Tif, Dtk, Etk-2) and 4E-BP3 (eIF4E-binding protein 3) have been selected from the cDNA library of human mammalian cell line by a Yeast-two Hybrid method. Tyro 3, a member of Axl/Sky/Mer RTKs (Receptor Tyrosin kinase) family, plays an important role in cell growth, differentiation, and metabolism. In this study, RPA2 interacts with Tyro 3 were demonstrated by the Yeast-two Hybrid and immunoprecipitation methods, indicating that RPA2 may participate in Tyro 3-dependent cell growth, differentiation or metabolism. Another putative RPA2-binding protein, 4E-BP3, is a member of 4E-BPs family. It is a 14.6 kDa protein that is able to compete with eIF4G for binding to eIF4E (eukaryotic initiation factor 4E), thereby stifling eIF4F complex-directed translation. In this study, RPA2 interacts with 4E-BP3 was confirmed by immunoprecipitation assay. The truncated form of RPA2 lacking N-terminal, middle domain, or C-terminal was construced and expressed by TnT quick coupled transcription/translation kit. The result of immunoprecipitation assay indicated that 4E-BP3 interacted with full-length RPA2 and with RPA2 lacking c-terminal domain (amino acid 170-270) suggesting that the binding area of RPA2 with 4E-BP3 located within phosphorylation domain and DNA-binding domain of RPA2. Our previous studies found that RPA2-4E-BP3 complex may dissociate in the UV-stimulated cells. In the study, cell lysates of HEK293 were prepared at different times after UV irradiation with 50J/m2, separated into nuclear and cytoplasmic fractions, and the amounts of RPA2 protein in cellular fractions were determined by western blot analysis. The results of this experiment revealed that protein levels of RPA2 in both nuclear and cytoplasmic fractions remain constent after UV irradiation. In summary, results of this study demonstrated RPA2 interacted with 4E-BP3 or Tyro 3. After UV damage, the interaction of RPA2 and 4E-BP3 disappeared. This situation is not aroused from the change of protein level of RPA2. In addition, this study also showed that the binding region of RPA2 with 4E-BP3 located within phosphorylation domain and DNA-binding domain of RPA2.

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