Table_2_In vitro DNA Inversions Mediated by the PsrA Site-Specific Tyrosine Recombinase of Streptococcus pneumoniae.DOCX

Site-specific recombination is a DNA breaking and reconstructing process that plays important roles in various cellular pathways for both prokaryotes and eukaryotes. This process requires a site-specific recombinase and direct or inverted repeats. Some tyrosine site-specific recombinases catalyze DNA inversions and regulate subpopulation diversity and phase variation in many bacterial species. In Streptococcus pneumoniae, the PsrA tyrosine recombinase was shown to control DNA inversions in the three DNA methyltransferase hsdS genes of the type I restriction-modification cod locus. Such DNA inversions are mediated by three inverted repeats (IR1, IR2, and IR3). In this work, we purified an untagged form of the PsrA protein and studied its DNA-binding and catalytic features. Gel retardation assays showed that PsrA binds to linear and supercoiled DNAs, containing or not inverted repeats. Nevertheless, DNase I footprinting assays showed that, on linear DNAs, PsrA has a preference for sites that include an IR1 sequence (IR1.1 or IR1.2) and its boundary sequences. Furthermore, on supercoiled DNAs, PsrA was able to generate DNA inversions between specific inverted repeats (IR1, IR2, and IR3), which supports its ability to locate specific target sites. Unlike other site-specific recombinases, PsrA showed reliance on magnesium ions for efficient catalysis of IR1-mediated DNA inversions. We discuss that PsrA might find its specific binding sites on the bacterial genome by a mechanism that involves transitory non-specific interactions between protein and DNA.

位点特异性重组（site-specific recombination）是一类DNA断裂与重接过程，在原核生物与真核生物的多种细胞通路中发挥关键作用。该过程需要位点特异性重组酶（site-specific recombinase）以及正向或反向重复序列参与。部分酪氨酸位点特异性重组酶（tyrosine site-specific recombinase）能够催化DNA倒位，并在多种细菌物种中调控亚群多样性与相变异（phase variation）。
在肺炎链球菌（Streptococcus pneumoniae）中，研究已证实PsrA酪氨酸重组酶可调控I型限制修饰编码基因座（type I restriction-modification cod locus）内3个DNA甲基转移酶（DNA methyltransferase）hsdS基因的DNA倒位。此类DNA倒位由3组反向重复序列（inverted repeat，IR1、IR2与IR3）介导。
本研究中，我们纯化获得了无标签形式的PsrA蛋白，并对其DNA结合与催化特性展开系统研究。凝胶阻滞实验（gel retardation assay）结果显示，PsrA可结合带有或不带有反向重复序列的线性DNA与超螺旋DNA（supercoiled DNA）。然而，DNase I足迹实验（DNase I footprinting assay）结果表明，在线性DNA底物上，PsrA对包含IR1序列（IR1.1或IR1.2）及其侧翼边界序列的位点具有结合偏好性。此外，在超螺旋DNA底物上，PsrA可介导特定反向重复序列（IR1、IR2与IR3）之间发生DNA倒位，这证实其能够精准识别特异性靶位点。与其他位点特异性重组酶不同，PsrA的IR1介导的DNA倒位高效催化活性依赖镁离子的存在。
本研究讨论认为，PsrA或可通过一种涉及蛋白与DNA之间短暂非特异性相互作用的机制，在细菌基因组中定位其特异性结合位点。