Molecular architecture of the preinitiation complex in adenovirus DNA replication

After infection of a host cell, adenovirus (Ad) aims for generation of progeny viruses, and thus it rapidly replicates its genomic DNA. The replication process starts with the assembly of the preinitiation complex (PIC) on the origin DNA. The PIC consists of three viral proteins, DNA polymerase (pol), precursor terminal protein (pTP), DNA binding protein (DBP) and two transcription factors of the host cell, Nuclear Factor I (NFI) and Octamer binding protein (Oct-1). Both transcription factors enhance the replication process up to 80 fold. The efficiency of Ad DNA replication is an essential issue for gene therapy procedures, and because most human cell types contain the cellular transcription factors NFI and Oct-1, it is important to study how NFI and Oct-1 perform their function. One of the mechanisms by which NFI and Oct-1 could stimulate replication is by changing the structure of the origin DNA. Furthermore, NFI and Oct-1 recruit pol and pTP to the core origin through direct protein-protein interactions. Experiments in Chapter 2 and Chapter 3 of this thesis investigate the first mechanism. The ability of NFI and Oct-1 to induce a DNA bend in the Ad5 origin is analyzed using scanning force microscopy (SFM). Chapter 2 demonstrates that NFI induces a 60° bend upon binding to the origin DNA, which is functionally important. Mutations of the A/T-rich region preceding the NFI recognition sequence in the origin reduce the bend angle and lead to a loss of NFI-stimulated replication. Chapter 3 shows that Oct-1 induces a 42° bend in the origin DNA. Moreover, simultaneous binding of NFI and Oct-1 to the origin increases the collective DNA bend up to 82°, which indicates that the two DNA bends induced individually by NFI and Oct-1 are oriented towards each other. Consequently, such extensive DNA bending leads to the synergistic stimulation of replication. Furthermore, the Addendum presents that, in contrast to NFI, mutation of the A/T-rich region does not effect the Oct-1-induced DNA bend and its ability to stimulate replication. All these data generate a model, in which bending of the origin DNA by NFI and Oct-1 facilitates the formation of an optimal nucleoprotein structure of the PIC.
After PIC formation, pol initiates Ad DNA replication using pTP as a primer. Both proteins form a tight heterodimer, the pTP/pol complex, which plays a central role in initiation. During this process pol covalently couples the first nucleotide of a new viral genome to the hydroxyl group of the Ser580 residue of pTP that is positioned in the active site of pol. Protein priming is a unique system to start DNA replication, and therefore it is important to understand its mechanism. The priming region of pTP that surrounds Ser580 contains a number of negatively charged amino acids (13 aa out of 43), and since the protein primer and the DNA primer bind to a common site of pol, it is likely that pTP mimics DNA in order to interact with pol. Chapter 4 demonstrates that the conserved acidic residues of pTP, Asp578 and Asp582, located close to Ser580 are important for the optimal initiation of Ad DNA replication. Moreover, they contribute to the kinetics of initiation, showing that these residues play an important role in the arrangement of an optimally functioning active site of pol that leads to an efficient initiation. This also suggests that pTP is not a simple primer but rather an integral part of the initiating pTP/pol complex. The Addendum demonstrates that also other conserved acidic residues from the priming region, Glu585 and Asp594, located somewhat further from Ser580 are important for the initiation. This suggests that the optimal positioning of the Ser580 hydroxyl group in the active site of pol is facilitated by charge-charge interactions between pTP and pol. In Chapter 5, a model of the PIC assembly and protein priming is proposed and future perspectives for the Ad5 DNA replication are discussed.