粒線體DNA (mtDNA) 的完整性對粒線體的氧化磷酸化系統—即製造能量的功能，相當重要。受損的mtDNA需要即時地被修復或清除，否則累積的基因缺陷會導致粒線體功能失常，並發展出各式疾病。有關此胞器如何應付mtDNA損傷的詳細機制至今仍未被清楚闡明。研究指出，雙股斷裂損傷的mtDNA 會在數小時內迅速被降解，並此降解過程是由粒線體DNA 聚合酶γ (DNA polymerase subunit gamma, POLG)、粒線體基因組維持外切核酸酶1 (Mitochondrial Genome Maintenance Exonuclease 1; MGME1)、以及粒線體DNA解旋酶 (Twinkle; TWNK) 負責。有趣的是，這三個酵素同時也負責mtDNA的複製。在目前提出的mtDNA降解模型中，斷裂的mtDNA的其中一股會由POLG的3′-外切酶活性負責降解，另一股則由MGME1的5′-外切酶活性降解。TWNK則負責將雙股DNA解旋，以提供兩外切酶單股DNA受質。在此，我們將此三個酵素組成的複合體命名為粒線體基因組降解體。從分子結構的觀點，此降解體如何組裝並執行其功能，引起了我們實驗室的高度興趣。因此，在本研究中，我們首先透過蛋白質共表達，嘗試利用親和性液相層析來共純化此三個蛋白。然而，於體外，TWNK的溶解度低，並且不易形成正確的聚合化結構，此兩種情形皆不利於蛋白複合體的組裝，因此共表達的方法並未成功。接著，我們轉而將這三個蛋白分別純化，再將其混合藉此重建蛋白複合體。我們也設計了一DNA受質，並將其加入此複合體的組裝。目前我們正在嘗試優化組裝此蛋白複合體的條件。一旦取得此複合體，我們會使用負染電子顯微檢視此複合體的品質，並使用單分子冷凍電子顯微進行分子結構的解析。我們預期此研究將闡明mtDNA降解的結構機制，並增進我們對此基因組品質控管系統的了解。

The integrity of mitochondrial DNA (mtDNA) is crucial for the function of oxidative phosphorylation system in mitochondria. Damaged mtDNA molecules need to be timely repaired or eliminated, or the accumulated genomic defects could cause mitochondrial dysfunction and lead to disease development. Mechanism about how the organelle deals with severe mtDNA damages, especially DNA double-stranded break (DSB), is largely unknown. It is known that DSB-damaged mtDNA will be rapidly degraded in hours. Surprisingly, the degradation process is mediated by a machinery consisting of mitochondrial DNA polymerase γ (POLG), mitochondrial genome maintenance exonuclease 1 (MGME1) and mitochondrial DNA helicase Twinkle (TWNK)—the three enzymes known for synthesizing mtDNA. In the proposed model, linearized mtDNA fragments are degraded simultaneously from one end, with one strand being degraded in 3′-to-5′ direction via the 3′-exonuclease activity of POLG; and the other strand being degraded in 5′-to-3′ direction by MGME1. The helicase TWNK goes ahead the two exonucleases to unwind double-stranded DNA for degradation. How the three enzymes assemble to compose the mtDNA degradation machinery is unclear. Aiming to reveal the molecular structure of the machinery, here, we are trying to reconstitute the complex consisting of human POLγ, TWNK and MGME1 in vitro. We firstly tried co-expressing the three enzymes in insect cells. By tagging PolgA (the catalytic subunit of POLG holoenzyme), we aimed to co-purify the complex through affinity purification. However, the strategy did not success, owing to the poor solubility of TWNK and its heterogenous oligomerization state in vitro—both would harm protein complex formation. Therefore, we went for the secondary strategy, in which we purified the three proteins to homogenous individually and tried to reconstitute the complex by mixing them together. We also designed a hairpin DNA to mimic degradation substrate for the complex reconstitution. Analytical size-exclusion chromatography was used to purify the protein-DNA complex. We are currently optimizing complex reconstitution condition. Once we obtain the complex, we will employ single-particle Cryo-EM for structural determination. We anticipate our result will elucidate the structural mechanism of mtDNA degradation and further our understanding about the quality control system of mtDNA.

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