Luis Enrique Donate

Large T-Antigen Double Hexamers Imaged at the Simian Virus 40 Origin of Replication

ABSTRACT The initial step of simian virus 40 (SV40) DNA replication is the binding of the large tumor antigen (T-Ag) to the SV40 core origin. In the presence of Mg2+ and ATP, T-Ag forms a double-hexamer complex covering the complete core origin. By using electron microscopy and negative staining, we visualized for the first time T-Ag double hexamers bound to the SV40 origin. Image processing of side views of these nucleoprotein complexes revealed bilobed particles 24 nm long and 8 to 12 nm wide, which indicates that the two T-Ag hexamers are oriented head to head. Taking into account all of the biochemical data known on the T-Ag–DNA interactions at the replication origin, we present a model in which the DNA passes through the inner channel of both hexamers. In addition, we describe a previously undetected structural domain of the T-Ag hexamer and thereby amend the previously published dimensions of the T-Ag hexamer. This domain we have determined to be the DNA-binding domain of T-Ag.

The DnaB·DnaC complex: a structure based on dimers assembled around an occluded channel

Replicative helicases are motor proteins that unwind DNA at replication forks. Escherichia coli DnaB is the best characterized member of this family of enzymes. We present the 26 Å resolution three‐dimensional structure of the DnaB hexamer in complex with its loading partner, DnaC, obtained from cryo‐electron microscopy. Analysis of the volume brings insight into the elaborate way the two proteins interact, and provides a structural basis for control of the symmetry state and inactivation of the helicase by DnaC. The complex is arranged on the basis of interactions among DnaC and DnaB dimers. DnaC monomers are observed for the first time to arrange as three dumb‐bell‐shaped dimers that interlock into one of the faces of the helicase. This could be responsible for the freezing of DnaB in a C3 architecture by its loading partner. The central channel of the helicase is almost occluded near the end opposite to DnaC, such that even single‐stranded DNA could not pass through. We propose that the DnaB N‐terminal domain is located at this face.

Large T antigen on the simian virus 40 origin of replication: a 3D snapshot prior to DNA replication

Large T antigen is the replicative helicase of simian virus 40. Its specific binding to the origin of replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three‐dimensional structure of the full‐length T antigen double hexamer assembled at its origin of replication by cryoelectron microscopy and single‐particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer–hexamer junction mainly at two opposite sites. The structure of the T antigen at the origin of replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA replication is proposed.

Bacteriophage T3 connector: Three-dimensional structure and comparison with other viral head-tail connecting regions☆

The bacteriophage T3 connector, which consists of 12 copies of protein gp8, has been studied by image processing of electron micrographs from negatively stained ordered aggregates. A three-dimensional reconstruction of T3 connectors was obtained by collection of tilted views and using the direct Fourier method, up to 2.3 nm resolution. The reconstructed unit cell contains two connectors whose main structural features are essentially identical, but facing in opposite directions. The T3 connector has a height of about 10.9 nm, with two clearly defined domains: a wider one 14.4 nm in diameter, with 12 morphological units in the periphery, and a narrower one, 9.7 nm in diameter. There is a channel clearly defined in the narrower domain that almost closes along the wider domain. Comparison of the three-dimensional structure obtained for the connector of phages T3 and phi 29, and that of the neck extracted from phage phi 29 particles, reveals striking similarities and significant differences. A model for a general connector to account for the common functions carried out by these viral assemblies is discussed together with the possible role of the channel for DNA translocation.

Production of λ-φ29 phage chimeras

Abstract Proheads of bacteriophage λA which carry the connector of phage φ29 instead of that of λ have been produced in vitro . These hybrid proheads have a structure similar to that of normal λ proheads. Furthermore, the chimeric proheads can package both λ and φ29 DNA. These data show that the connector domains involved in both head assembly and DNA packaging are functionally similar. The DNA-containing λ-φ29 proheads can be complemented in vitro with φ29 tails to yield infective particles capable of DNA transfer.

Characterization of a versatile in vitro DNA-packaging system based on hybrid λ/φ29 proheads

Abstract We have studied the assembly of bacteriophage λ head proteins on the phage φ29 connector to produce in vitro chimeric proheads, whose ability to package different types of DNA depends on the physical integrity of the φ29 connector. Terminal protein-free φ29 DNA as well as nonviral DNAs have been shown to be efficiently packaged by this hybrid system. An RNA, that can be provided by any of the extracts used in the complementation mixture, was required for DNA packaging, both by the hybrid system as well as by the homologous λ system. The DNA-packaging activity of RNase-treated proheads can be restored by adding a mixture of ribosomal RNAs. There is also a requirement for a minimal length of DNA to be stably packaged. The packaging protein pt 6 of φ29 can replace the A terminase complex in the in vitro packaging system, both with the chimeric as well as genuine λ proheads.