P Element Transposition in Vitro Proceeds by a Cut-And-Paste Mechanism and Uses GTP as a Cofactor

Cell. 1992 Apr 3;69(1):27-39. doi: 10.1016/0092-8674(92)90116-t.

Abstract

We have developed an in vitro reaction system for Drosophila P element transposition. Transposition products were recovered by selection in E. coli, and contained simple P element insertions flanked by 8 bp target site duplications as observed in vivo. Transposition required Mg+2 and partially purified P element transposase. Unlike other DNA rearrangement reactions, P element transposition in vitro used GTP as a cofactor; deoxyGTP, dideoxyGTP, or the nonhydrolyzable GTP analogs GMP-PNP or GMP-PCP were also used. Transposon DNA molecules cleaved at the P element termini were able to transpose, but those lacking 3'-hydroxyl groups were inactive. These biochemical data are consistent with genetic data suggesting that P element transposition occurs via a "cut-and-paste" mechanism.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • DNA Transposable Elements / physiology*
  • Drosophila melanogaster / genetics*
  • Escherichia coli / genetics
  • Guanosine Triphosphate / metabolism*
  • Magnesium / metabolism*
  • Molecular Sequence Data
  • Nucleotidyltransferases / metabolism*
  • Plasmids / genetics
  • Temperature
  • Transposases

Substances

  • DNA Transposable Elements
  • Guanosine Triphosphate
  • Nucleotidyltransferases
  • Transposases
  • Magnesium