鏈黴菌中ParA1蛋白參與染色體分配的 相關功能性區域之探討

Mapping functional domains responsible for DNA association and dynamic movement in ParA1 of Streptomyces coelicolor

巫冠毅(Kuan-Yi Wu)

指導教授 ： 史有伶

國立臺灣大學/生命科學院/生化科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.02193

摘要

The Par system為細菌體內參與將複製後的遺傳物質平均分配到子細胞中的一種機制。第一型Par system是由兩個蛋白質(ParA 與 ParB)，及類似著絲點的核甘酸序列parS所組成。ParA蛋白屬於the Walker type ATPase中的MinD/ParA蛋白質家族中的一員。此類蛋白質大多能藉蛋白質間的聚合與解離而在細菌內進行反覆的動態運動，以提供物理力量支援相關細胞功能；因而被認為擁有細胞骨架蛋白的基本特性。本研究之主要目的為利用鏈黴菌(S. coelicolor A3(2))在產孢過程中的染色體分配機制為研究對象，探討其ParA相似蛋白ParA1如何利用其動態細胞骨架特性，幫助細菌內染色體的分配。首先，我們將帶有黃色螢光蛋白標記的ParA1融合蛋白於大腸桿菌中表現，並在螢光顯微鏡下，觀察此融合蛋白在細菌內的動態運動現象與分佈狀態。在大腸桿菌內缺少ParB的狀態下，我們發現Yfp-ParA1融合蛋白分佈在大腸桿菌類核體(nucleoid)的位置，並觀察到Yfp-ParA1融合蛋白在類核體上能進行不規則的動態分佈。當ParA1胺端區域的第1-32個胺基酸被移除後，Yfp-ParA133-308融合蛋白的分佈狀況及動態運動散佈於細胞質內發生，而不再侷限在類核體上。我們也發現，不論是否有完整的胺端區域，ParA1融合蛋白都能於大腸桿菌中形成螺旋狀纖維構造；且此螺旋狀構造的出現與大腸桿菌中內生的細胞骨架系統the Min system與肌動蛋白類蛋白MreB均無相關性。這些結果支持ParA1的動態特質可單純透過ParA1本身產生。
我們進一步根據已知的ParA相似蛋白結構，進行一系列的點突變實驗來尋找ParA1不同功能性區域與其定位於類核體之間的關係。結果顯示，ParA1完整的ATP結合區域，為附著在細菌的類核體所需。突變胺端胺基酸Arg31，也會影響ParA1和類核體的結合。羧基端則有兩個胺基酸R218與R247參與類核體的附著。另一方面，我們也利用細菌雙雜交系統測試點突變後的ParA1與ParA1及其與ParB產生交互作用的能力，發現ParA1之間的交互作用需要正常的ATP結合區域參與；而在破壞ParA1中預測為與水解ATP相關的Asp68後，其與ParB的交互作用則更
加穩定。因此，ParA1內不同的功能性區域應以ATP結合區域為中心，藉著各類區域的特性使ParA1具有不同的功能。在將miniF上的質體分配系統置換成鏈黴菌的Par system後，發現鏈黴菌染色體的分配系統能將miniF穩定的分配到子細胞中，也直接證明了鏈黴菌的Par system是有主動分配遺傳物質的能力。
綜合以上結果，我們發現鏈黴菌ParA1蛋白的細胞骨架性質是建立在三個功能性區域的基礎上，不同功能性區域間會互相影響，形塑ParA1獨特的運動性質與在細菌內的分佈。而ATP的結合區域可能扮演調節ParA1與類核體或與ParB間交互作用的角色。這些特性推測與鏈黴菌產孢過程中的染色體分配機制有重要的關係。

關鍵字

the Par system ； 鏈黴菌 ； 細胞骨架 ； Walker型ATP水解酶

並列摘要

Chromosome segregation during sporulation in Streptomyces coelicolor A(3)2 is mediated by the Par system. The Par system is consisted of two protein components, ParA1 and ParB, and a centromere-like site, parS. ParA1 belongs to the MinD/ParA subgroup of the Walker-type ATPase family that is characterized by its cytoskeleton properties. Our goal is to gain better understanding on how the Par system may couple its dynamic cytoskeleton property to chromosome segregation. We show here that when expressed in Escherichia coli cells, Yfp tagged ParA1 in the absence of ParB was capable of associating with nucleoids and underwent stochastic movement over the nucleoids. On the other hand, the N-terminal truncated ParA1 preserved its ability to undergo stochastic movement though no longer associated with nucleoids. Both the full-length and N-terminal truncated ParA1 proteins were capable of forming long-range helical cables in E. coli that was independent of actin-like MreB and the Min system. Thus the dynamic movement of ParA1 is independent of ParB and can be separated from nucleoid association. Furthermore, ParA1’s ability to associate with nucleoids was impaired when mutations (R31E, K39E, G40V, K44E, D154A) were introduced into the ATP-binding pocket, suggesting that ATP binding to ParA1 is required for association with nucleoids. Using bacterial two-hybrid assays, ParA1-ParB interaction was enhanced when ParA1 harbored a mutation in Asp68, a residue that is predicted to be involved in ATP hydrolysis. This result indicates that ATP-bound ParA1 may stabilize its interaction with ParB. We also substitute the miniF Par system, SopABC, with Scoe Par system and found that Scoe Par system play active roles in plasmid DNA partition. In summary, we have characterized the functional domains of ParA1 of S. coelicolor A(3)2 that are fundamental for its cytoskeleton properties: (1) the N-terminal domain together with the ATP-binding pocket are responsible for targeting ParA1 to the bacterial nucleoids; (2) the ATP-bound ParA1 is likely to confer the ParB-binding conformation, and (3) the N-terminal domain of ParA1 is not required to deliver the dynamic properties of the protein.

並列關鍵字

chromosome segregation ； the Par system ； ParA ； ParB ； parS ； cytoskeleton ； Walker type ATPase

參考文獻

Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., et al. (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141-147.

Adam, M., Fraipont, C., Rhazi, N., Nguyen-Disteche, M., Lakaye, B., Frere, J.M., Devreese, B., Van Beeumen, J., van Heijenoort, Y., van Heijenoort, J., et al. (1997). The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate. J Bacteriol 179, 6005-6009.

Anand, S.P., Akhtar, P., Tinsley, E., Watkins, S.C., and Khan, S.A. (2008). GTP-dependent polymerization of the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis. Mol Microbiol 67, 881-890.

Autret, S., and Errington, J. (2003). A role for division-site-selection protein MinD in regulation of internucleoid jumping of Soj (ParA) protein in Bacillus subtilis. Mol Microbiol 47, 159-169.

Autret, S., Nair, R., and Errington, J. (2001). Genetic analysis of the chromosome segregation protein Spo0J of Bacillus subtilis: evidence for separate domains involved in DNA binding and interactions with Soj protein. Mol Microbiol 41, 743-755.