Hostname: page-component-78c5997874-j824f Total loading time: 0 Render date: 2024-11-08T01:54:08.955Z Has data issue: false hasContentIssue false
  • English
  • Français

Study of the VirE2-ssT-DNA Complex Formation by Scanning Probe Microscopy and Gel Electrophoresis— T-Complex Visualization

Published online by Cambridge University Press:  18 January 2007

Irina Volokhina  and
Mikhail Chumakov
Irina Volokhina
Affiliation:
Agrobacterial Transformation and Biotechnology Laboratory, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia Saratov State University, Faculty of Nonlinear Processes, Biophysics Department, 150 Ulitsa Moskovskaya, Saratov 410006, Russia
Mikhail Chumakov
Affiliation:
Agrobacterial Transformation and Biotechnology Laboratory, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia Saratov State University, Faculty of Nonlinear Processes, Biophysics Department, 150 Ulitsa Moskovskaya, Saratov 410006, Russia
Get access

Abstract

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 λ) 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.

Keywords

Agrobacterium tumefaciensAFMvirulence protein VirE2T-complex
Type
Research Article
Information
Microscopy and Microanalysis , Volume 13 , Issue 1: Special Issue: Environmental, Industrial, and AppliedMicrobiology , February 2007 , pp. 51 - 54
Copyright
2007 Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Abu-Arish, A., Frenkiel-Krispin, D., Fricke, T., Tzfira, T., Citovsky, V., Wolf, S.G. & Elbaum, M. (2004). Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA. J Biol Chem 279, 25359–25363.Google Scholar
Chrysogelos, S. & Griffith, J. (1982). Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units. Proc Natl Acad Sci USA 79, 5803–5807.Google Scholar
Chumakov, M.I. (2000). Transfer of genetic information from agrobacterial to bacterial and plant cells: Membrane and supramembrane structures involving in the transfer (an overview). Memb Cell Biol 13, 309–331.Google Scholar
Citovsky, V., Guralnik, B., Simon, M. & Wall, J.S. (1997). The molecular structure of Agrobacterium VirE2-single stranded DNA complexes involved in nuclear import. J Mol Biol 272, 718–727.Google Scholar
Gulyamov, M.O. & Jaminsky, I.V. (1998). Atomic Force Microscopy of Nucleic Acid. Moscow, Russia: Centre for Prospective Technologies, Physics Department, Moscow State University (in Russian).
Jefferson, R.A., Burgess, S.M. & Hirsh, D. (1986). Beta-glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83, 8447–8451.Google Scholar
Velikov, V.A., Ermoshina, O.S., Volokhina, I.V. & Chumakov, M.I. (2006). Production of miniantibodies to Agrobacterium tumefaciens VirE2 virulence protein by the method of phage display. Mol Gen Mikrobiol Virusol 1, 21–24 (in Russian).Google Scholar
Volokhina, I.V., Sazonova, I.A., Velikov, V.A. & Chumakov, M.I. (2005). Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens. Microbiol Res 160, 67–73.Google Scholar