thesis

The role of the adenovirus DNA binding protein in DNA replication and recombination

Abstract

Replication of adenovirus DNA in infected cells is an efficient process that, compared to cellular replication, has the use of a protein primer as a hallmark. The mechanism of this DNA replication process and especially the role of one of the replication proteins, the DNA binding protein DBP, is the main subject of this thesis. Adenovirus DNA replication can be reconstituted in vitro, using three viral proteins, adenovirus DNA polymerase (pol), precursor terminal protein (pTP), and DBP. Optimal replication efficiency is obtained when two cellular transcription factors are added, nuclear factor I (NFI) and Oct-1. The adenoviral dsDNA genome contains two terminal proteins (TP) covalently linked to the 5. ends. The inverted terminal repeats contain the origins of replication. pTP and pol are tightly associated in solution. During initiation of replication pTP functions as a primer to which the first nucleotide, dCTP, is covalently coupled. Both NFI and Oct-1 stimulate the initiation by recruiting the pTP-pol complex to the origin of replication. Initiation starts opposite position 4 of the template strand. After formation of a pTP-trinucleotide (pTP-CAT), the complex jumps back and CAT becomes paired with template residues 1.3. Shortly after jumping back, the polymerase dissociates from pTP and elongation proceeds via strand displacement. The adenovirus DNA binding protein is an important player in adenovirus DNA replication, where it serves multiple functions. In the first step of adenovirus DNA replication DBP stimulates the coupling of the first nucleotide to pTP. Also binding of NFI to the origin is stimulated by DBP. Subsequently, during elongation DBP unwinds the dsDNA ahead of pol and removes secondary structures. Adenovirus DBP binds with high affinity and cooperativity to ssDNA, whereas binding to dsDNA is non- cooperative and with lower affinity. These differences in binding affinity are the driving force for dsDNA unwinding which is required for processive DNA chain elongation by pol. DBP is a 529 amino acids long protein with a molecular weight of 59 kD. The structure of adenovirus DBP possesses a remarkable feature; the C-terminal arm (aa. 512-529), which is important for the cooperative binding of DBP to another DBP molecule. In addition, the C-terminal arm is flexible and can rotate around a fixed point, called the hinge-region (aa. 512-515). In Chapter 1, an introduction to the adenovirus DNA replication machinery and the role of several single stranded DNA binding proteins originating from different organisms is presented. In Chapter 2 we present data on the function of the flexibility of the C-terminal arm of DBP and we discuss the implications of an altered flexibility on adenovirus DNA replication by mutating several aa in the hinge-region. In Chapter 3, we present data on the function of DBP in the stimulation of initiation. In contrast to a direct protein-protein interaction of DBP with pol we demonstrate that DBP stimulates the binding of pol to the dsDNA origin. We assume that the structure of the dsDNA template is changed by DBP in such a way that polymerase binds more efficiently to the dsDNA. In Chapter 4 we demonstrate that DBP functions in unwinding of the dsDNA donor and that it has an additional role in renaturation of the unwound donor with the complementary acceptor ssDNA. For the first time we were able to demonstrate a direct role for an adenovirus replication protein in homologous recombination. Finally, in Chapter 5 new questions, that have surfaced as a result of the research presented in this thesis, are discussed

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