Anatomy of a flexer-DNA complex inside a higher-order transposition intermediate.
Escherichia coli HU, a nonsequence-specific histone- and HMG-like DNA-binding protein, was chemically converted into a series of HU-nucleases with an iron-EDTA-based cleavage moiety positioned at 16 rationally selected sites. Specific DNA cleavage patterns from each of these HU-nucleases allowed us to determine the precise localization, stoichiometry, and orientation of HU binding in the Mu transpososome, a multiprotein structure that mediates the chemical reactions in DNA transposition. Correlation of the DNA cleavage data with the position of the cleavage moiety in the HU three-dimensional structure indicates the presence of a dramatic DNA bend, for which the bend center, direction, and magnitude were assessed. The data, which directly localize selected HU amino acids with respect to DNA in the transpososome, were used as constraints for computer-based molecular modeling to derive the first snapshot of an HU-DNA interaction.
Lavoie
B D
BD
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
Shaw
G S
GS
Millner
A
A
Chaconas
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D019895:Transposases
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C040330:histone-like protein HU, bacteria; D004254:DNA Nucleotidyltransferases; D019895:Transposases