Esnault Charles, Jaillet Jérôme, Delorme Nicolas, Bouchet Nicolas, Renault Sylvaine, Douziech-Eyrolles Laurence, Pilard Jean-François, Augé-Gouillou Corinne
Groupe Instabilité Génétique et Transposases, EA 6306, Fédération GICC, UFR Sciences Pharmaceutiques, Université François Rabelais, 31 Avenue Monge, 37200 Tours (France).
Chembiochem. 2015 Jan 2;16(1):140-8. doi: 10.1002/cbic.201402466. Epub 2014 Dec 8.
Transposases are specific DNA-binding proteins that promote the mobility of discrete DNA segments. We used a combination of physicochemical approaches to describe the association of MOS1 (an eukaryotic transposase) with its specific target DNA, an event corresponding to the first steps of the transposition cycle. Because the kinetic constants of the reaction are still unknown, we aimed to determine them by using quartz crystal microbalance on two sources of recombinant MOS1: one produced in insect cells and the other produced in bacteria. The prokaryotic-expressed MOS1 showed no cooperativity and displayed a Kd of about 300 nM. In contrast, the eukaryotic-expressed MOS1 generated a cooperative system, with a lower Kd (∼ 2 nm). The origins of these differences were investigated by IR spectroscopy and AFM imaging. Both support the conclusion that prokaryotic- and eukaryotic-expressed MOS1 are not similarly folded, thereby resulting in differences in the early steps of transposition.
转座酶是一类特定的DNA结合蛋白，可促进离散DNA片段的移动。我们运用多种物理化学方法来描述MOS1（一种真核转座酶）与其特定靶DNA的结合，这一事件对应于转座循环的起始步骤。由于该反应的动力学常数仍未知，我们旨在通过石英晶体微天平对两种重组MOS1来源进行测定：一种在昆虫细胞中产生，另一种在细菌中产生。原核表达的MOS1未表现出协同性，其解离常数（Kd）约为300 nM。相比之下，真核表达的MOS1形成了一个协同体系，Kd较低（约2 nM）。通过红外光谱和原子力显微镜成像研究了这些差异的根源。两者均支持以下结论：原核和真核表达的MOS1折叠方式不同，从而导致转座早期步骤存在差异。
