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.