Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.
Virulence proteins VirD1 and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirD1-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirD1 and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirD1 protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirD1, fails to nick linear double-stranded DNA templates in vitro.
Jasper
F
F
Max-Planck-Institut für Züchtungsforschung, Köln, Germany.
Koncz
C
C
Schell
J
J
Steinbiss
H H
HH
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D012231:Rhizobium / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D037521:Virulence Factors
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; C036483:T-DNA; D037521:Virulence Factors; D004706:Endodeoxyribonucleases