Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition.
Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.
Izsvák
Zsuzsanna
Z
Max Delbrück Center for Molecular Medicine, Robert Rössle Strasse 10, D-13092 Berlin, Germany.
Khare
Dheeraj
D
Behlke
Joachim
J
Heinemann
Udo
U
Plasterk
Ronald H
RH
Ivics
Zoltán
Z
D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D019895:Transposases / Q000737:chemistry
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D004247:DNA; C081454:Tc1 transposase; D019895:Transposases