The replication of DNA is an intricate process that requires the coordinated action of many components. Defects in replication can lead to catastrophic consequences for an organism, so the acquisition of in-depth knowledge of the mechanism and components of DNA replication has direct application to solving problems related to human health. Since the mechanisms of DNA replication are conserved among all biological entities, the relatively simplicity of the replication machinery of bacteriophage T7 makes it an ideal model system to study mechanistic aspects of DNA replication. DNA primase plays a crucial role in DNA replication because DNA polymerases cannot initiate DNA chains de novo. This essential class of enzymes catalyzes the synthesis of ribonucleotides that serve as primers for DNA polymerase for the initiation of Okazaki fragment synthesis. DNA primase is also involved in loading of DNA helicase, in the regulation of replication, and handoff of the primer to DNA polymerase. The objective of this proposal is to investigate three major aspects of T7 DNA primase function that are currently obscure employing biophysical and biochemical methods. Specifically, the aims of this proposal are to study the molecular bases for the regulation of primer synthesis and handoff by bacteriophage T7 DNA primase through the investigation of: the role of primase subdomain interactions in primer synthesis (Aim 1), the role conserved amino acid in the stabilization of the primer-template duplex (Aim 2), and the process of primer handoff to DNA polymerase (Aim 3). The results obtained from these studies will provide insight into fundamental functions of DNA primase and should be broadly applicable to other replication systems.
The replication of DNA is an essential process of all living things. DNA primases are enzymes that are critical for the initiation of replication and lagging- strand synthesis;in addition they play central regulatory roles in the coordination of the replication fork. We will exploit the relative simplicity of the bacteriophage T7 DNA replication system to investigate the mechanisms of primer synthesis and handoff by these enzymes.
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