STRUCTURES OF LARGE T-ANTIGEN AT THE ORIGIN OF SV40 DNA-REPLICATION BY ATOMIC-FORCE MICROSCOPY

For inorganic crystals such as calcite (CaCO3), Atomic Force Microscop y (AFM) has provided surface structure at atomic resolution (Ohnesorge and Binnig, 1993). As part of a broad effort to obtain high resolutio n for an individual protein or protein assembly (Binnig et al., 1986; Rugar and Hansma, 1990; Radmacher et al., 1992), we applied AFM to stu dy the ATP-dependent double hexamer of SV40 large T antigen, which ass embles around the viral origin of DNA replication. Multimeric mass has been determined in two-dimensional projected images by Scanning Trans mission 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 hei ght by AFM or as a fraction of mass thickness by STEM, is of comparabl e magnitude. On either side of the cleft, hexamers attain a maximum he ight 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 o f complete and partial replication assemblies consistent with the spat ial distribution of recognition pentanucleotides on the DNA, and they contribute direct geometrical evidence for a ring-like hexamer structu re.