Tuesday, April 1, 2025
4/1/2025
DNA Replication Machines: Structure-Function Studies - Project Summary/Abstract Our laboratory is involved in structure-function studies of DNA replication. Over the next five years we propose to carry out two related projects, one focusing on human DNA replication and the other on replication of herpes simplex virus type 1 (HSV1) DNA. Knowledge gained from these projects about the structural basis of the fundamental processes enabling precise replication of human genome and replication of viral DNA will provide a foundation for the development of novel therapies to treat a wide variety of diseases. Project 1. High-fidelity genome replication is the foundation of healthy life. The bulk of human DNA replication is performed by the B-family DNA polymerases. Primase-polymerase α (Primosome) synthesizes chimeric RNA-DNA primers, before switching them to Polε and to Polδ for the start of leading and lagging strands replication, respectively. Despite recent progress in structural studies of B-family DNA polymerases, significant gaps remain in our knowledge regarding the mechanisms of their function. This incomplete understanding warrants additional study, especially of the determinants that tightly coordinate polymerase transactions at the replication fork. Consequently, revealing step by step the details of human DNA replication events and the coordinated action of the involved proteins remains a key project in our laboratory and is the major focus of the current project. Recently, based on the initial structural and functional characterization of Primosome, we proposed a new direction of study examining how the C-terminal domain of the primase large subunit, along with a chimeric RNA-DNA primer, fulfills the role of global regulator of transactions at the replication fork. Here, we will expand these studies using multiple approaches to characterize human Primosome transactions. Project 2. The human herpesvirus (HHV) family includes eight members grouped into α, β, and γ subfamilies, which cause a variety of diseases. The replisomes of all HHVs require six essential components: a single- strand DNA-binding protein, a two-subunit DNA polymerase complex, and a three-subunit helicase-primase (HP) complex. Approved therapies for HHV infections mainly target inhibition of the polymerase subunit, but the HP complex is an even more attractive target since its DNA unwinding and primer synthesis functions precede the processive polymerization. The search for HP inhibitors (HPIs) is ongoing, but success has been limited to α-HHVs. Improvement of current HPIs and the development of novel HPIs targeting the members of the β and γ subfamilies are complicated by the lack of knowledge about HP structures and function, as well as the detailed mechanism of inhibition. HP is an attractive target for comparative analysis with its human counterpart, since the DNA unwinding and primer formation by HP subunits occur in a significantly compact complex and requires precise coordination of both activities. Here, we propose to commence studies of HHV replication with comprehensive structure-function analysis of HSV1 HP and its mechanism of inhibition.
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