Flanigan Asa, Gardner Jeffrey
Department of Microbiology, University of Illinois in Urbana-Champaign, Urbana, Illinois, USA.
BMC Microbiol. 2008 Nov 17;8:199. doi: 10.1186/1471-2180-8-199.
The Gifsy-I phage integrates into the Salmonella Typhimurium chromosome via an integrase mediated, site-specific recombination mechanism. Excision of the Gifsy-I phage requires three proteins, the Gifsy-I integrase (Int), the Gifsy-I excisionase (Xis) protein, and host encoded Integration Host Factor (IHF). The Gifsy-I xis gene encodes the 94-residue Gifsy-I excisionase protein that has a molecular weight of 11.2 kDa and a pI of 10.2. Electrophoretic Mobility Shift Assays (EMSA) suggested at least one region of the protein is responsible for protein-DNA interactions with a tripartite DNA binding site composed of three direct imperfect repeats.
Here we have undertaken experiments to dissect and model the structural motifs of Gifsy-I Xis necessary for its observed DNA binding activity. Diethyl sulfate mutagenesis (DES) and mutagenic PCR techniques were used to generate Gifsy-I xis mutants. Mutant Xis proteins that lacked activity in vivo were purified and tested by EMSA for binding to the Gifsy-I Xis attP attachment site. Results from mutagenesis experiments and EMSA were compared to results of structural predictions and sequence analyses.
Sequence comparisons revealed evidence for three distinct structural motifs in the Gifsy-I Xis protein. Multiple sequence alignments revealed unexpected homologies between the Gifsy-I Xis protein and two distinct subsets of polynucleotide binding proteins. Our data may suggest a role for the Gifsy-I Xis in the regulation of the Gifsy-I phage excision beyond that of DNA binding and possible interactions with the Gifsy-I Int protein.
Gifsy-I噬菌体通过整合酶介导的位点特异性重组机制整合到鼠伤寒沙门氏菌染色体中。Gifsy-I噬菌体的切除需要三种蛋白质，即Gifsy-I整合酶（Int）、Gifsy-I切除酶（Xis）蛋白和宿主编码的整合宿主因子（IHF）。Gifsy-I xis基因编码94个氨基酸的Gifsy-I切除酶蛋白，其分子量为11.2 kDa，pI为10.2。电泳迁移率变动分析（EMSA）表明，该蛋白至少有一个区域负责与由三个直接不完全重复序列组成的三方DNA结合位点进行蛋白质-DNA相互作用。
在这里，我们进行了实验，以剖析和模拟Gifsy-I Xis观察到的DNA结合活性所必需的结构基序。使用硫酸二乙酯诱变（DES）和诱变PCR技术产生Gifsy-I xis突变体。纯化在体内缺乏活性的突变Xis蛋白，并通过EMSA测试其与Gifsy-I Xis attP附着位点的结合。将诱变实验和EMSA的结果与结构预测和序列分析的结果进行比较。
序列比较揭示了Gifsy-I Xis蛋白中三个不同结构基序的证据。多序列比对揭示了Gifsy-I Xis蛋白与两个不同的多核苷酸结合蛋白亚群之间意外的同源性。我们的数据可能表明，Gifsy-I Xis在Gifsy-I噬菌体切除的调控中所起的作用，超出了DNA结合以及与Gifsy-I Int蛋白可能的相互作用。
