The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity.
The bacterial insertion sequence IS911 transposes via a covalently closed circular intermediate. Circle formation involves transposase-mediated pairing of both insertion sequence ends. While full-length transposase, OrfAB, binds poorly in vitro to IS911 DNA fragments carrying a copy of the IS911 end, truncated protein derivatives carrying the first 135 (OrfAB[1-135]) or 149 (OrfAB[1-149]) amino acid residues bind efficiently. They generate a paired-end complex containing two such fragments which resembles that expected for the first synaptic complex. Shorter protein derivatives lacking a region involved in multimerisation do not form these complexes but modify the binding of OrfAB[1-135] and OrfAB[1-149]. DNaseI footprinting demonstrated that OrfAB[1-149] protects a sub-terminal (internal) region of the inverted repeats which includes two blocks of sequence (beta and gamma) conserved between the left (IRL) and right (IRR) ends. DNA binding assays in vitro and measurement of recombination activity in vivo of sequential deletion derivatives of the two inverted repeats suggested a model in which the N-terminal region of OrfAB binds the conserved boxes beta and gamma in a sequence-specific manner and anchors the two insertion sequence ends into a paired-end complex. The external region of the inverted repeat is proposed to contact the C-terminal transposase domain carrying the catalytic site.
Normand
C
C
Laboratoire de Microbiologie et Génétique Moléculaire, CNRS, 118 Route de Narbonne, Toulouse, 31062, France.
Duval-Valentin
G
G
Haren
L
L
Chandler
M
M
D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D008957:Models, Genetic; D016366:Open Reading Frames / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D020079:Terminal Repeat Sequences / Q000235:genetics*; D019895:Transposases / Q000737:chemistry / Q000378:metabolism*
D015342:DNA Probes; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D019895:Transposases; D003850:Deoxyribonuclease I