title: Kinetic analysis of the interaction of Mos1 transposase with its inverted terminal repeats reveals new insight into the protein-DNA complex assembly.
abstract: 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.
authors.affiliation: Groupe Instabilité Génétique et Transposases, EA 6306, Fédération GICC, UFR Sciences Pharmaceutiques, Université François Rabelais, 31 Avenue Monge, 37200 Tours (France).
authors.forename: Charles
authors.initials: C
authors.lastname: Esnault
authors.forename: Jérôme
authors.initials: J
authors.lastname: Jaillet
authors.forename: Nicolas
authors.initials: N
authors.lastname: Delorme
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bouchet
authors.forename: Sylvaine
authors.initials: S
authors.lastname: Renault
authors.forename: Laurence
authors.initials: L
authors.lastname: Douziech-Eyrolles
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Pilard
authors.forename: Corinne
authors.initials: C
authors.lastname: Augé-Gouillou
mesh_terms: D000818:Animals; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004330:Drosophila / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D058525:Quartz Crystal Microbalance Techniques; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D020079:Terminal Repeat Sequences; D019895:Transposases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D019476:Insect Proteins; D011994:Recombinant Proteins; C111652:mariner transposases; D004247:DNA; D019895:Transposases
keywords: DNA cleavage; biochemical constants; mariner transposons; mobile DNA; protein structures; transposition