The terminal nucleotide of the Mu genome controls catalysis of DNA strand transfer.
Members of the transposase/retroviral-integrase superfamily use a single active site to perform at least two reactions during transposition of a DNA transposon or a retroviral cDNA. They hydrolyze a DNA sequence at the end of the mobile DNA and then join this DNA end to a target DNA (a reaction called DNA strand transfer). Critical to understanding the mechanism of recombination is elucidating how these distinct reactions are orchestrated by the same active site. Here we find that DNA substrates terminating in a dideoxynucleotide allow Mu transposase to hydrolyze a target DNA, combining aspects of both natural reactions. Analyses of the sequence preferences for target hydrolysis and of the structure of the cleaved product indicate that this reaction is promoted by the active site in the conformation that normally promotes DNA strand transfer. Dissecting the DNA requirements for target hydrolysis reveals that the ribose of the last nucleotide of the Mu DNA activates transposase's catalytic potential, even when this residue is not a direct chemical participant. These findings provide insight into the molecular mechanism insuring that DNA strand transfer ordinarily occurs rather than inappropriate DNA cleavage. The required presence of the terminal nucleotide in the transposase active site creates a great advantage for the attached 3'OH to serve as nucleophile.
Goldhaber-Gordon
Ilana
I
Department of Biology, Massachusetts Institute of Technology, and Howard Hughes Medical Institute, 77 Massachusetts Avenue, 68-523, Cambridge, MA 02139, USA.
Early
Michael H
MH
Baker
Tania A
TA
D010583:Bacteriophage mu / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000378:metabolism / Q000648:ultrastructure*; D016679:Genome, Viral; D006868:Hydrolysis; D008957:Models, Genetic; D011485:Protein Binding; D011995:Recombination, Genetic; D013997:Time Factors; D019895:Transposases / Q000378:metabolism
D004247:DNA; C114697:mu transposase; D019895:Transposases