In vitro recombination and inverted terminal repeat binding activities of the Mcmar1 transposase.
The Mcmar1 mariner element (MLE) presents some intriguing features with two large, perfectly conserved, 355 bp inverted terminal repeats (ITRs) containing two 28 bp direct repeats (DRs). The presence of a complete ORF in Mcmar1 makes it possible to explore the transposition of this unusual MLE. Mcmar1 transposase (MCMAR1) was purified, and in vitro transposition assays showed that it is able to promote ITR-dependent DNA cleavages and recombination events, which correspond to plasmid fusions and transpositions with imprecise ends. Further analyses indicated that MCMAR1 is able to interact with the 355 bp ITR through two DRs: the EDR (external DR) is a high-affinity binding site for MCMAR1, whereas the IDR (internal DR) is a low-affinity binding site. The main complex detected within the EDR contained a transposase dimer and only one DNA molecule. We hypothesize that the inability of MCMAR1 to promote precise in vitro transposition events could be due to mutations in its ORF sequence or to the specific features of transposase binding to the ITR. Indeed, the ITR region spanning from EDR to IDR resembles a MITE and could be bent by specific host factors. This suggests that the assembly of the transposition complex is more complex than that of those involved in the mobility of the Mos1 and Himar1 mariner elements.
Renault
Sylvaine
S
Université François Rabelais de Tours, GICC, CNRS, UMR 6239, UFR des Sciences & Techniques, Parc de Grandmont, 37200 Tours, France. sylvaine.renault@univ-tours.fr
Demattéi
Marie-Véronique
MV
Lahouassa
Hichem
H
Bigot
Yves
Y
Augé-Gouillou
Corinne
C
D001483:Base Sequence; D018983:DNA Footprinting; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D020079:Terminal Repeat Sequences / Q000235:genetics*; D019895:Transposases / Q000235:genetics* / Q000378:metabolism*
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D019895:Transposases