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(Received for publication, August 4,
1994; and in revised form, October 12, 1994) T-DNA processing during agroinfection of plants is initiated by
site- and strand-specific incision at the T-DNA border sequences of the
Ti plasmid. Two proteins are required for this reaction: VirD2 (49.6
kDa), catalyzing a site-specific cleaving-joining reaction on
single-stranded DNA in vitro (Pansegrau, W., Schoumacher, F.,
Hohn, B., and Lanka, E.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11538-11542), and VirD1 (16.1 kDa), an accessory protein
required for VirD2-mediated specific cleavage of double-stranded DNA.
Following efficient overproduction, VirD1 was isolated in active form
from inclusion bodies and purified to near homogeneity. The protein was
applied together with purified VirD2 protein for specific cleavage of
double-stranded T-DNA border sequences in vitro. The reaction
proceeds on negative superhelical DNA and requires Mg
Volume 270,
Number 3,
Issue of January 20, 1995 pp. 1269-1276
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
PURIFIED PROTEINS VirD1 AND VirD2 CATALYZE SITE- AND
STRAND-SPECIFIC CLEAVAGE OF SUPERHELICAL T-BORDER DNA IN VITRO
ions. Relaxed DNA is not cleaved. The 5` terminus of the broken
DNA strand is covalently associated with protein, most probably VirD2,
and the cleavage site is located at the same position that is found in vivo, indicating that the in vitro reaction mimics
the one that takes place in induced agrobacteria. Relaxation of plasmid
DNA occurs only upon addition of protein denaturants, suggesting that
the DNA in the VirD1/VirD2 complex is topologically constrained by
strong protein-DNA interactions. The characteristics of the
VirD1/VirD2-mediated cleavage reaction strongly resemble those observed
with relaxosomes of IncP plasmids involved in initiation of transfer
DNA replication during bacterial conjugation.
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