ENZYMATIC MECHANISMS OF DNA REPLICATION

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A working model for our studies is that assembly of a replication fork at a yeast chromosomal origin of replication occurs in multiple stages: recognition, unwinding, priming of DNA synthesis by DNA polymerase alpha - primase, and recruitment of pol delta and/or pol epsilon. In a complicated, multicomponent process such as DNA replication, even the most exhaustive characterization of the individual proteins cannot describe the dynamics of the actual physiological events. In order to do that, interaction between the components, no matter how many there are, must be studied. The experimental theme of this proposal is therefore to design both biochemical and genetic experiments to characterize the dynamic interactions between the replication proteins, especially the helicase that unwinds and the polymerases that form the primosome and elongation apparatus. Ultimately we would like to reconstitute an unwinding and initiation event at an origin with purified proteins. Study of the proteins described herein will provide essential background and materials for doing so. The proposal itself is divided into two parts, the first of which concerns unwinding. We have identified a new yeast mutant that affects a gene encoding a protein with DNA helicase activity. The specificity of the helicase suggests that it may be involved in the initiation of DNA replication and since such a helicase may form the linchpin of replication fork assembly, we will characterize the helicase and its interactions with the Origin Replication Complex, ORC, and the DNA polymerases. The second part of the proposal deals with further characterization of the three replication polymerases. The two least well characterized polymerases, pol delta and pol epsilon, will be emphasized. The goal is to isolate "holoenzymes" of each replicative DNA polymerase and then to investigate how they interact with each other physically and functionally. There is no paradigm in prokaryotic systems for two, much less three, genetically distinct polymerases interacting during DNA replication, so very basic studies are warranted. Monoclonal affinity purification of pol e will be carried out. Furthermore, the gene will be used to establish the function of the C-terminal portion of the protein, which may be involved in protein/protein interactions. For studies of pol delta, we will take advantage of our ability to express the catalytic subunit free of other subunits in E. coli to purify the 55 kDa subunit and determine its function. We will continue using the gene to characterize pol delta's interaction with PCNA, the critical protein that tethers the polymerase to the DNA. We propose that a helicase directs the polymerases to the fork and this forms a link between the two parts of the proposal.