中文摘要
DEDDh 3 端至5 端核酸水解外切酶家族的成員包含了RNA 水解酶以DNA 水
解酶兩種，並且廣泛的參與了各種RNA 生合成、代謝以及DNA 的降解、複製和修
復。然而此家族成員如何與核酸受質的3 端結合以及如何挑選目標受質等機制的相
關研究依然有所不足，所以我們挑選兩個DEDDh 核酸水解外切酶家族的成員
(CRN-4 與 RNase T) 做為我們研究的標的，更進一步的探討CRN-4 在DNA 降解
以及RNase T 在RNA 成熟時所扮演的角色。
CRN-4 (細胞凋亡相關水解酶4) 是秀麗桿狀線蟲中參與細胞凋亡的核酸水解酶
之一，想要了解CRN-4 如何參與在細胞凋亡的核酸水解階段，因此分析了CRN-4
的胞內與胞外的生化功能、並且解析了CRN-4 的晶體結構。研究指出在溶液以及晶
體結構中CRN-4 是以雙聚體的形式存在，並且在細胞凋亡的生理條件下具有最高的
水解活性，晶體結構與點突變的實驗也指出CRN-4 具有典型的DEDDh 核酸水解外
切酶活性中心，而位於C 端的鋅結合功能區則是帶有正電荷，可能負責與帶負電的
DNA 受質結合。整合所有實驗結果，我們推測CRN-4 在細胞凋亡時期利用C 端的
鋅結合功能區與DNA結合，再藉由N端的DEDDh 核酸水解外切酶功能區降解DNA
受質，達到細胞凋亡時的DNA 降解之目的。
RNase T 是細菌中的3 端至5 端核酸水解外切酶，廣泛的参與在5S、23S 核醣
體RNA 和轉錄RNA 等穩定的小型RNA 的生合成，其活性會受到RNA/DNA 3 端
的Cytosine 以及雙股的RNA/DNA 所抑制。我們發現非RNase T 所偏好的核酸
(C:Cytosine)出現在RNA 受質的3 端時，會導致RNase T 結構改變，也因此無法降
解3 端具有C 的RNA 受質；RNase T 的特殊雙聚體構型也會根據雙股的最後成對
的核酸為G-C 結合或U-A 結合而生成在3 端的2 或1 個單股核酸突出。研究結果
顯示RNase T 利用這兩種調控的方式，參與多種類的RNA 分子的3 端成熟過程，
產生不同種類的成熟RNA 分子，包含了tRNA、4.5S RNA、5S rRNA 以及23S rRNA。
總合來說，此論文提供了CRN-4 與 RNase T 詳細的生化功能以及晶體結構的
相關研究，RNase T 結構分析結果也提供了一個嶄新的DEDDh 3 端至5 端核酸水解
外切酶家族受質挑選、結合及降解的機制。有鑒於DEDDh 3 端至5 端核酸水解外
切酶家族成員的三級結構組成以及活性中心皆相似於CRN-4 與 RNase T，也因此
我們研究不但提供了DEDDh 3 端至5 端核酸水解外切酶家族結構上的組成與受質
挑選的相關機制，也給予了其他與疾病相關的DEDDh 核酸水解外切酶家族成員之
目標受質挑選及受質種類的研究上可依循的線索。

Abstract
The DEDD-family exonucleases are involved in various aspects of RNA processing and degradation, as well as DNA proofreading and repair. However, how this family of exonucleases binds at the 3'-end of a nucleic acid chain, and selects and digests their target substrates is mostly unknown. In this thesis, two DEDD-family exonucleases, CRN-4 and RNase T, have been selected for biochemical and structural studies to elucidate their functional roles in DNA degradation and RNA maturation, respectively.
CRN-4 (Cell death-related nuclease 4) was identified as one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. We analyzed CRN-4’s biochemical properties and in vivo cellular functions, and determined the crystal structures of CRN-4 in apo-form, Mn2+-bound active form, and Er3+-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn2+-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4’s function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis.
RNase T is a bacterial 3'-to-5' exonuclease involved in the final trimming of many stable RNA, including 5S and 23S ribosomal RNA and transfer RNA. The exonuclease activity of RNase T is blocked by a 3'-terminal cytosine and double-stranded structures. Our crystal structural analyses on four RNase T-DNA complexes show that a “C-filter” in RNase T screens out the nucleic acids with a 3'-terminal cytosine by inducing a disruptive conformational change at the active site. The two subunits of the RNase T dimer work together in binding a double-stranded structure, producing a minimum product of a duplex with a 2-nt or 1-nt 3' overhang, depending on the last base pair, G-C or U-A, in the duplex. Our results reveal the general principles and the underlying working mechanisms for the final trimming step made by RNase T in the maturation of ribosomal, transfer and small stable RNA.
In summary, this thesis presents the biochemical properties and crystal structures of two DEDD-family nucleases. The structural studies in RNase T reveal the underlying working mechanism of this enzyme in substrate selection and digestion. As many DEDD nucleases share a fold and a conserved active site similar to that of CRN-4 and RNase T, the structural insight provided in this study not only shows the link between the DEDD domain arrangement and substrate specificity, but also reveals clues to understand the exonuclease activity and to identify possible substrates of other DEDD-family proteins, some of which have been linked directly to human diseases.

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