粒線體帶有自己的基因組，稱為粒線體DNA (mtDNA)。此基因組負責製造粒腺體中氧化磷酸化系統所需要的關鍵蛋白質。因此，維持mtDNA的品質與完整性，對於粒線體的功能乃至於細胞的健康與存活相當重要。粒線體DNA解旋酶Twinkle為協助mtDNA複製與降解的主要DNA解旋酶，是維持mtDNA品質與完整性的一關鍵酵素。Twinkle屬於超家族解旋酶4 (superfamily helicase 4)其中的一員，這個家族的解旋酶多半會形成一個六聚體、高度協調的環狀結構，並能夠藉由水解ATP來驅動其N端和C端結構域的相互協調 (domain coordination)，因此得以在單股DNA上以特定的方向前進，過程中能將另一股DNA移除，以達到解旋雙股DNA的目的。目前已知發生在人類Twinkle上的許多點突變，會破壞此酵素結構域之間的協調性，因此干擾它的聚合並損害其酵素活性，最終導致系統性的粒線體疾病，如進行性外眼肌麻痺，以及粒線體DNA耗竭綜合症7等。最近發表的結構研究，揭示了突變體Twinkle-W315L的兩個原子解析度的模型，均為無DNA結合的八聚體和七聚體結構。然而目前對於野生型Twinkle的分子結構研究，雖然已成功揭示了此酵素家族典型的六聚體環狀結構，但因解析度較低，缺乏結構域之間相互作用的結構訊息。因此，Twinkle作用的結構機制仍是未知。在本研究中，我們的目標為解析人類Twinkle與DNA形成複合體的分子結構，以闡明此酵素在DNA上前進的結構機制。目前，透過分析級粒徑排阻層析法，我們已成功純化出均質的野生型Twinkle。在酵素活性測試中，純化的Twinkle也如預期的表現出ATP依賴性DNA解旋活性，以及與DNA結合的能力。為解析Twinkle與DNA結合的分子結構，我們設計了一髮夾(hairpin) DNA用以模擬複製叉的構型，將其與Twinkle混合形成複合體，並經過粒徑排阻層析法純化。我們將使用負染電子顯微鏡對此複合體進行初步分析。同時，我們也將利用X射線晶體學進行此複合體的結構分析。我們預計本項研究除可闡明Twinkle作用的結構機轉，還可進一步拓展我們對mtDNA維持機制的了解，或可對粒線體功能失常引起的相關疾病之治療，有所助益。

Mammalian mitochondria contain a circular genome called mitochondrial DNA (mtDNA) which encodes proteins necessary for the oxidative phosphorylation system (OXPHOS). The quality control of mtDNA, therefore, is crucial for maintaining mitochondrial function. Mitochondrial DNA helicase Twinkle plays a vital role in mtDNA maintenance by acting as the master helicase in both replication and degradation of the genome. The enzyme belongs to superfamily helicase 4 (SF4). This family of helicases oligomerize to form a highly coordinated, ring-like architecture. Their DNA unwinding activity relies on an ATP-driven coordination between their N-terminal and C-terminal domains which enables these helicases to translocate on DNA in a specific direction. In the case of Twinkle, residue substitutions that disrupt the domain coordination harm its activity, resulting in systemic mitochondrial diseases such as progressive external ophthalmoplegia (PEO) and mitochondrial DNA depletion syndrome 7 (MTDPS7). A recent published structural study of Twinkle uncovered two atomic-resolution models of a disease-causing variant (Twinkle-W315L), adopting DNA-free octameric and heptameric states. However, harmed by the structural heterogeneity of wild-type Twinkle, so far, its structural analysis only resulted in a cryo-EM map around 11.6 Å. The structure although successfully revealed a classical hexameric-ring architecture of SF4 helicases, the structural information regarding interplay with DNA and ATP-driven domain coordination, namely the molecular mechanism of the enzyme’s action, is still lacking. In the present study, we are aiming to dissect the molecular structure of human Twinkle in complex with DNA. To this aim, recombinant Twinkle protein has been successfully purified to homogeneous. After a two-step polishing by analytical size-exclusion chromatography (SEC), we have successfully obtained pure, active wild-type Twinkle exhibiting DNA-binding and ATP-stimulated DNA unwinding activities. By in vitro complex assembly, we have putatively obtained the complex of Twinkle bound with a forked hairpin DNA and cofactors as revealed by SEC. The sample will soon be subjected to negative-stain EM for analysis. In the meanwhile, crystallization screen of the Twinkle-DNA complex will also be conducted. Taking together, we anticipate our study will uncover the structural basis of Twinkle’s action and extend our understanding about the molecular mechanism of mtDNA maintenance.

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