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.