參與單純疱疹病毒一型UL12切割核酸功能的胺基酸之探究; Roles of conserved charged amino acid residues of herpes simplex virus typeⅠ UL12 in nuclease activities

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Title:	參與單純疱疹病毒一型UL12切割核酸功能的胺基酸之探究;Roles of conserved charged amino acid residues of herpes simplex virus typeⅠ UL12 in nuclease activities
Authors:	陳奕瑾;Yi-Jin Chen
Contributors:	中國醫藥大學：醫學研究所碩士班
Keywords:	單純疱;疹病毒;核酸水解酶;nuclease;herpes;UL12
Date:	2007-07-11
Issue Date:	2009-08-13 14:50:37 (UTC+8)
Abstract:	單純疱疹病毒一型 (Herpes simplex virus type 1, HSV1) 的UL12是一種鹼性核酸水解酶，擁有明顯的Exonuclease活性以及較微弱的Endouclease活性。UL12目前被研究出與病毒基因體的成熟與包裝有著密切關係，另外，也發現UL12參與病毒基因體複製且催化DNA股的置換最後形成基因重組的現象。為進一步分析HSV-1 UL12催化此現象的機制，我們以pET載體攜帶UL12基因，並利用大腸桿菌系統表現UL12。經由大腸桿菌表現出來的UL12在pH值為10，鎂離子濃度為3 mM擁有較佳的酵素活性，之後，我們將疱疹病毒各代表物種之UL12胺基酸序列進行Multiple alignment，選出19個高度保留且帶電荷的鹼性與酸性胺基酸，將這19個胺基酸以定點突變的方式置換成alanine，並利用大腸桿菌系統表現蛋白質，進一步分析比較這些突變株對於核酸水解功能、與DNA結合的能力以及鎂離子需求濃度上有何不同。在核酸水解功能的分析結果中，突變株R235A、R496A、R531A、K366A、N494A、D329、E280A、E364A的核酸水解能力是顯著降低的。更進一步分析，突變株R496A、R531A、K366A、N494A、D329A與DNA結合有關；突變株R235A、E280A、E364A與鎂離子結合相關。藉由以上的實驗結果利用程式模擬出其3D蛋白質結構，模擬出的UL12與E. coli DNA polymeraseⅡ的exonuclease domain相似度最高，並藉由此結構推測出對於HSV-1 UL12而言，催化水解DNA的機制似乎與E. coli DNA polymeraseⅡ的exonuclease domain是相似的。 Herpes simplex virus type 1 (HSV-1) UL12 is an alkaline nuclease with a strong exonuclease activity and a weak endonuclease activity. UL12 is involved in the maturation and packaging of viral genomic DNA. It also catalyzes the strand exchange and promotes the viral genetic recombination. To analyze the catalytic mechanism of HSV-1 UL12, we expressed UL12 in Escherichia coli with the use of a pET expression vector and purified to homogeneity. HSV-1 UL12 exhibited an alkaline preference with a pH optimum of 10.0. It also requires magnesium ions with a magnesium optimum of 3 mM. Multiple alignment analysis showed that 19 charged amino acid residues were highly conserved among herpesviruses. We therefore replaced the charged amino acid residues with alanine by site-directed mutagenesis, and the nuclease activities, DNA-binding abilities, and magnesium requirements of UL12 mutants were analyzed. Mutations at Arg-235, Arg-496, Arg-531, Lys-366, Asn-494, Asp-329, Glu-280, and Glu-364 showed drastically reduced nuclease activities. Mutations at Arg-235, Glu-280, and Glu-364 were closely related to bind of magnesium ion. Homology modeling analysis showed that the proposed structure of HSV-1 UL12 was similar to the nuclease domain of E. coli DNA polymeraseⅡ. These findings suggested that HSV-1 UL12 might follow a similar catalytic mechanism as the nuclease domain of E. coli DNA polymeraseⅡ.
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