Structural and biochemical studies of large T antigen: the SV40 replicative helicase

Simian Virus 40 (SV40) replication has long been regarded as a useful model system in circumventing the complexity of studying the eukaryotic DNA replication process. SV40 large T antigen (LTag), as the only virus-encoding protein required for viral genome replication, extensively uses cellular proteins to function as a replication initiator at replication origins and as a helicase during nascent DNA elongation. ❧ During replication initiation, DNA polymerase alpha-primase (pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. At least three subunits of pol-prim interact physically with the hexameric SV40 LTag to carry out its functions. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. ❧ Further DNA elongation substantially depends on the helicase function of LTag. In the presence of DNA, LTag forms an efficient molecular motor fueled by ATP binding and hydrolysis. Significant progress has been made in gaining insight into the mechanism of LTag helicase function by structural and biochemical studies. However, the detailed mechanism by which LTag couple the ATP hydrolysis to translocation and DNA separation is not yet clear. ❧ This dissertation is organized to further the understanding of these areas via a literature review and presentation of findings I discovered through my PhD research: ❧ In Chapter 1, I will give a thorough review about the current understanding of LTag mediated SV40 replication. ❧ In Chapter 2, I will present a co-crystal structure of SV40 hexameric helicase and the regulatory subunit (p68) of eukaryotic DNA polymerase ɑ/primase (pol/prim). The structure reveals the detailed LTag-p68 interface, which is validated by site-directed mutagenesis, and demonstrated to be critical in activating the SV40 primosome in cell-free reactions with purified pol-prim, as well as in monkey cells in vivo. ❧ In Chapter 3, I will demonstrate the roles of the residues along the LTag central channel structure elements in DNA unwinding. By substituting these residues with a series of amino acids carrying diverse side chain properties and systematically examining the DNA stimulated ATPase activity and helicase function of these mutants, my study reveals the significant roles of these central channel residues in DNA unwinding. More intriguingly, careful data analysis suggests that even though these residues are spatially proximal to each other, they might affect DNA unwinding through different mechanisms. ❧ Chapter 4 describes the detailed experimental procedures. ❧ Last but not the least, efforts and preliminary results are included in the appendices towards understanding of structure and function of the complete LTag protein, as well as its tumorigenesis through interacting with human retinoblastoma protein. ❧ Altogether, the information presented here advances the understanding of the mechanism of SV40 LTag in DNA replication and provides a solid base for future studies with this incredible molecule. We anticipate results accumulated in this model system will eventually facilitate the understanding of the replication process as well as tumorigenesis in eukaryotic cells.