Substrate recognition and induced DNA deformation by transposase at the target-capture stage of Tn10 transposition.
The bacterial transposon Tn10 inserts preferentially into sites that conform to a 9 bp consensus sequence: 5' NGCTNAGCN 3'. However, this sequence is not on its own sufficient to confer target specificity as the base-pairs flanking this sequence also contribute significantly to target-site selection. We have performed a series of "contact-probing experiments" to define directly the protein-DNA interactions that govern target-site selection in the Tn10 system. The HisG1 hotspot for Tn10 insertion was the main focus here. We infer that there is a rather broad zone ( approximately 24 bp) of contact between transposase and target DNA in the target-capture complex. This includes base-specific contacts at all of the purine residues in the consensus positions of the target core and primarily backbone contacts out to 7-8 bp in the two flanking regions immediately adjacent to the core. Also, highly localized sites of chemical hypersensitivity are identified that reveal symmetrically disposed deformations in DNA structure in the target-capture complex. Furthermore, the level of strand transfer is shown to be reduced by phosphorothioate substitution of phosphate groups at or close to the sites of target DNA deformation. Interestingly, for one particular target DNA, a mutant form of HisG1 called MutF, the above phosphorothioate inhibition of strand transfer is suppressed by replacing Mg(2+) with Mn(2+). Based on these results a model for sequence-specific target capture is proposed which attempts to define possible relationships between transposase interactions with the target core and flanking sequences, transposase-induced DNA deformation of the target site and divalent metal ion binding to the target-capture complex.
Pribil
P A
PA
Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Haniford
D B
DB
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence / Q000235:genetics; D019327:Copper Sulfate; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D019175:DNA Methylation; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005038:Ethylnitrosourea; D008345:Manganese / Q000494:pharmacology; D008957:Models, Genetic; D008958:Models, Molecular; D009690:Nucleic Acid Conformation / Q000187:drug effects; D010618:Phenanthrolines; D011995:Recombination, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013379:Substrate Specificity / Q000187:drug effects; D013463:Sulfuric Acid Esters; D013873:Thionucleotides / Q000235:genetics / Q000378:metabolism; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D010618:Phenanthrolines; D013463:Sulfuric Acid Esters; D013873:Thionucleotides; D008345:Manganese; D004247:DNA; D019895:Transposases; C007482:dimethyl sulfate; D019327:Copper Sulfate; D005038:Ethylnitrosourea; C025205:1,10-phenanthroline