Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.
The bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA bending.
Pribil
Patrick A
PA
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Wardle
Simon J
SJ
Haniford
David B
DB
D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004251:DNA Transposable Elements / Q000502:physiology*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis; D011995:Recombination, Genetic; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013873:Thionucleotides / Q000235:genetics / Q000378:metabolism; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D009841:Oligonucleotides; D013873:Thionucleotides; D019895:Transposases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D002118:Calcium