Study of the VirE2-ssT-DNA complex formation by scanning probe microscopy and gel electrophoresis- T-complex visualization.
The recombinant virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into plant cells, was isolated, purified, and used for interactions with ssT-DNA. The in vitro interaction of VirE2 and ss-binding protein from Escherichia coli with single-stranded DNA (phage lambda) was determined by agarose gel electrophoresis by the formation of high-molecular-weight complexes after preliminary coincubation of purified protein preparations with ssDNA. We show that VirE2 binds to single-stranded DNA and protects it against nuclease S1 degradation much better than does E. coli SSB protein. We for first time observed the VirE2-ssT-DNA complex by using atomic force microscopy. The complex observed by atomic force microscopy after ssT-DNA and VirE2 protein mixing has a length of about 800 nm and a 5-8 nm width in sites with attached VirE2 protein.
Volokhina
Irina
I
Agrobacterial Transformation and Biotechnology Laboratory, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia.
Chumakov
Mikhail
M
D016960:Agrobacterium tumefaciens / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods*; D029968:Escherichia coli Proteins / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D020527:Microscopy, Scanning Probe / Q000379:methods*; D011485:Protein Binding; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; C051583:virE2 protein, Agrobacterium; D015719:Single-Strand Specific DNA and RNA Endonucleases