假性狂犬病毒(Pseudorabies virus)是引起假性狂犬病的病因，為?疹病毒科的一員。假性狂犬病毒DNA結合蛋白(DNA-binding protein)已被證實與病毒DNA複製和基因體重組(recombination)有關。在本研究中，我們進一步分析假性狂犬病毒DNA結合蛋白在同源性重組(homologous recombination)的關鍵步驟-strand invasion-的過程中所扮演的角色。假性狂犬病毒DNA結合蛋白可以促進strand invasion，使一段單股DNA插入同源性的質體中，形成D loop的結構。假性狂犬病毒DNA結合蛋白催化strand invasion的反應是發生在中性的環境中，而且反應過程中需要鎂離子的協助，在鎂離子濃度為20 mM時是最佳的反應條件。進一步以無水醋酸進行化學修飾後，發現離胺酸殘基在假性狂犬病毒DNA結合蛋白形成D loop結構時扮演重要的角色。經過胺基酸序列比對之後，我們發現在假性狂犬病毒DNA結合蛋白的23個離胺酸(lysine)殘基中有3個離胺酸殘基具有高度保留性，其所在位置分別在第161、756及970個胺基酸上。因此我們將上述離胺酸殘基置換成丙氨酸(alanine)殘基。實驗結果顯示，假性狂犬病毒DNA結合蛋白中的第756個位置的離胺酸與其DNA結合功能有關，而第161個位置的離胺酸則和strand invasion功能有關。我們更進一步的使用假性狂犬病毒DNA結合蛋白系列缺損株(N端缺損株與C端缺損株)分析參與strand invasion的功能區，結果顯示假性狂犬病毒DNA結合蛋白與strand invasion有關的功能區域極可能位於第1~199個胺基酸的區域，而位於第161個位置的離胺酸則可能是假性狂犬病毒DNA結合蛋白strand invasion的關鍵胺基酸。; Pseudorabies virus (PRV), the causative agent of pseudorabies, is a member of Herpesviridae. Herpesviral DNA-binding protein (DBP) is a viral protein involved in viral DNA replication and genomic recombination. In this study, we analyzed the role of DBP in strand invasion, a key event of homologous recombination. PRV DBP was able to promote the assimilation of a single-stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop (D loop). DBP catalyzed strand invasion in a neutral condition and in a magnesium-dependent manner with an optimum at 20 mM. Acetic anhydride modification assay revealed that lysine residues were important for D-loop formation of DBP. By comparison of amino acid sequences of herpesviral DBP homologs, three out of 23 lysine residues in PRV DBP, corresponding to Lys-161, Lys-756, and Lys-970, were completely conserved in all herpesviral DBPs. We therefore replaced these lysine residues by alanine residues. Data showed that Lys-756 of PRV DBP was involved in DNA-binding ability and Lys-161 of PRV DBP was involved in strand invasion ability. We further analyzed the functional domain of PRV DBP involved in strand invasion by nested-deletion mutants. Data showed that the region spanning 1~199 amino acid residues of PRV DBP was important for strand invasion. Taken together, the results indicated that N-terminal region ( 1~199 amino acid residues ) of PRV DBP was important for strand invasion and Lys-161 might be the key residue involved in strand invasion.
