Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro.
Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.
Ziemienowicz
A
A
Friedrich Miescher-Institut, CH-4002 Basel, Switzerland. ziemien@biotech.univ.gda.pl
Tinland
B
B
Bryant
J
J
Gloeckler
V
V
Hohn
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011088:DNA Ligases / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D018390:Gene Targeting; D008957:Models, Genetic; D009841:Oligonucleotides / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology; D010936:Plant Extracts / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000502:physiology*; D010947:Plants, Toxic; D011995:Recombination, Genetic; D013942:Thymine Nucleotides / Q000494:pharmacology; D013997:Time Factors; D014026:Nicotiana / Q000201:enzymology; D037521:Virulence Factors
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D009841:Oligonucleotides; D010936:Plant Extracts; D010940:Plant Proteins; D013942:Thymine Nucleotides; D037521:Virulence Factors; D000255:Adenosine Triphosphate; D011088:DNA Ligases; C024157:thymidine 5'-triphosphate