Structures of large T antigen at the origin of SV40 DNA replication by atomic force microscopy

• Published in: Biophysical journal Vol. 66; no. 2; pp. 293 - 298
• Main Authors: Mastrangelo, I.A., Bezanilla, M., Hansma, P.K., Hough, P.V., Hansma, H.G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.1994
• Subjects: Antigens, Viral, Tumor - chemistry; Antigens, Viral, Tumor - ultrastructure; Biophysical Phenomena; Biophysics; DNA Replication; Microscopy - methods; Microscopy, Electron, Scanning Transmission; Molecular Structure; Protein Conformation; Simian virus 40 - chemistry; Simian virus 40 - immunology
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(94)80800-8

For inorganic crystals such as calcite (CaCO3), Atomic Force Microscopy (AFM) has provided surface structure at atomic resolution (Ohnesorge and Binnig, 1993). As part of a broad effort to obtain high resolution for an individual protein or protein assembly (Binnig et al., 1986; Rugar and Hansma, 1990; Radmacher et al., 1992), we applied AFM to study the ATP-dependent double hexamer of SV40 large T antigen, which assembles around the viral origin of DNA replication. Multimeric mass has been determined in two-dimensional projected images by Scanning Transmission Electron Microscopy (STEM) (Mastrangelo et al., 1989). By AFM, if the DNA-protein preparation has been stained positively by uranyl acetate, the contour at the junction between hexamers is visible as a cleft, 2–4 nm deep. The cleft, whether determined as a fraction of height by AFM or as a fraction of mass thickness by STEM, is of comparable magnitude. On either side of the cleft, hexamers attain a maximum height of 13–16 nm. Monomers found in the absence of ATP show heights of 5–7 nm. Taken together, the z coordinates provide a surface profile of complete and partial replication assemblies consistent with the spatial distribution of recognition pentanucleotides on the DNA, and they contribute direct geometrical evidence for a ring-like hexamer structure.