Project Details


The regulatory control of DNA replication and cell proliferation is one
of the most fundamental processes in the life of an organism. Loss of
cells's responsiveness to normal mechanisms of regulation results in
cancerous growth of cells. The regulation of eukaryotic DNA replication
is a complex process involving many protein-protein and protein-DNA
interactions at various points. Among these are the commitment of cells
to enter the S phase of the cell cycle, which is sensitive to a number
of environmental influences such as the growth-stimulating factors and
addition of serum. Such stimulation results in a pleiotypic response of
events leading to DNA replication. One such response of cells is a
marked increase in the level of the dinucleotide, diadenosine
tetraphosphate, Ap4A. Ap4A is involved in multiple cellular events such
as DNA replication and cell proliferation, DNA repair and platelet
aggregation and vascular tonus. A protein that specifically binds to
Ap4A has been detected in various prokaryotic and eukaryotic cells and
we have found that in HeLa cells, this binding protein is part of a
multiprotein DNA polymerase complex that is capable of replicating SV40
DNA in vitro. As part of our long-term goal of understanding the
regulation of DNA replication in eukaryotes at the molecular level, we
have proposed to study the physiological role of the Ap4A binding
protein during DNA replication in this proposal. In the duration of
this project, we will purify the binding protein to homogeneity,
characterize the binding protein regarding its physical and biochemical
properties, determine the role of this protein in SV40 in vitro DNA
replication and the ability of Ap4A and the binding protein in
initiating DNA replication. We will determine the amino acid
composition and sequence of the binding protein and generate polyclonal
and monoclonal antibodies to this protein. Utilizing these reagents, we
will study the expression of this protein during various phases of the
cell cycle and the intracellular location of this protein. Making use
of the amino acid sequence data, we will generate oligonucleotide probes
and clone the cDNA for the binding protein. In continuation this
project, we will perform experiments to define the pbysiological role of
this binding protein in the various cellular events that Ap4A modulates.
Effective start/end date6/07/9130/06/95


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