Assembly and Mechanism of Action of Sulfolobus solfataricus DNA Replication Complexes

DNA replication enzymes are essential for the maintenance and propagation of genetic information which precisely governs the growth and development of our cells. Aberrant DNA replication processes have been implicated in a wide variety of human diseases, most notably cancer, and therefore, mechanistic understanding of DNA replication processes is paramount for the development of human therapeutic agents. The study of the eukaryotic replication system however, is difficult, as the system contains a large number of enzymes and regulatory factors making assembly of these systems for in vitro study complicated. Thus, in order to gain insight into the workings of the eukaryotic replication system, several model systems are used, where the complexity of the replication pathways is not as great.\ud \ud The DNA replication system from the thermophilic archaeon Sulfolobus solfataricus is a recently identified model with components sharing high levels of sequence homology to their eukaryotic counterparts. This system is ideal for gaining insight into the mechanistic workings of DNA replication which can be translated to the eukaryotic system. A key advantage to the study of thermophilic enzymes is in the ability to utilize reaction temperatures far lower than the physiological conditions for the organisms. This results in slower kinetics with no significant change in overall function, allowing an easier discernment of the enzyme’s mechanistic details. \ud \ud I have contributed to the development of Sulfolobus solfataricus as a model system primarily through characterization of nucleotide transferase enzymes including DNA polymerases and primases. Firstly, I have determined that the DNA polymerase, SsoPolB3, possesses a low rate of synthesis and fidelity more similar to those involved in lesion bypass. Secondly, I characterized the assembly and mechanism of action SsoPolB1 replication holoenzyme which replicates in a distributive fashion similar to the eukaryotic Pold holoenzyme, and maintains stimulated replication rates through rapid re-recruitment of the polymerase to the processivity clamp. Finally, I discovered and characterized the interactions of a unique primosome complex formed between the bacterial like DnaG primase and eukaryotic like MCM helicase. In all, my thesis provides for a more thorough understanding of the interactions, kinetics, and dynamics occurring at the replication fork.\u