title: Structures of large T antigen at the origin of SV40 DNA replication by atomic force microscopy.
abstract: 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.
authors.affiliation: Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
authors.forename: I A
authors.initials: IA
authors.lastname: Mastrangelo
authors.forename: M
authors.initials: M
authors.lastname: Bezanilla
authors.forename: P K
authors.initials: PK
authors.lastname: Hansma
authors.forename: P V
authors.initials: PV
authors.lastname: Hough
authors.forename: H G
authors.initials: HG
authors.lastname: Hansma
mesh_terms: D000957:Antigens, Viral, Tumor / Q000737:chemistry* / Q000648:ultrastructure; D055592:Biophysical Phenomena; D001703:Biophysics; D004261:DNA Replication; D008853:Microscopy / Q000379:methods; D017348:Microscopy, Electron, Scanning Transmission; D015394:Molecular Structure; D011487:Protein Conformation; D013539:Simian virus 40 / Q000737:chemistry / Q000276:immunology*
chemical_list: D000957:Antigens, Viral, Tumor