Targeting of a functional Escherichia coli RecA protein to the nucleus of plant cells.
We have characterised a RecA protein fused to the simian virus 40 large T nuclear-localisation signal. The fusion protein was targeted to the nucleus in transgenic tobacco plants with high efficiency. By contrast, authentic RecA was not enriched in the nuclei of plant cells expressing comparable amounts of protein. For detailed characterisation of the strand-exchange activity of the nuclear-targeted RecA protein, a nearly identical protein was expressed in Escherichia coli and purified to homogeneity. This protein was found to bind to single-stranded DNA with the same stoichiometry and to promote the exchange of homologous DNA strands with the same kinetics as authentic RecA. It was concluded that the amino-terminal modification did not alter any of the essential properties of RecA and that the fusion protein is a fully functional strand-exchange protein. However, the ATPase activity of this protein was 20 times greater than that of RecA in the absence of single-stranded DNA. As with RecA, this activity was further stimulated by the addition of single-stranded DNA. Since ATPase activity is correlated with the ability of RecA to assume its high affinity state for DNA, the nuclear-targeted RecA protein might be regarded as a constitutively stimulated RecA variant, fully functional in promoting homologous recombination.
Reiss
B
B
Max-Planck-Institut für Züchtungsforschung, Koln, Germany.
Kosak
H
H
Klemm
M
M
Schell
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases