ENZYMATIC MECHANISMS OF DNA REPLICATION

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A working model for our studies is that initiation of DNA replication at a yeast chromosomal origin of replication occurs in multiple stages: unwinding initiation of DNA synthesis by DNA polymerase alpha, and fork assembly with pol delta and/or pol epsilon. The proposal, itself, is divided into two parts, the first of which concerns the DNA polymerases. There are three essential DNA polymerases in yeast, pol alpha, pol delta and pol epsilon, and both pol alpha and pol delta are required for DNA replication. We must resolve one open question by demonstrating whether the essential function of pol epsilon is in replication or repair. The nature of our approach to polymerase studies will then change from past emphasis on reverse genetics on the catalytic core subunits to more biochemical approaches. We will focus increasingly on the role of pol (alpha, because of its apparently central role in coupling unwinding and fork assembly. The long term goal is to isolate holoenzymes of each replicative DNA polymerase and then to investigate how they interact with each other on a physical basis and how each one stimulates the other in the various model reactiolis catalyzed by DNA polymerases. 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. The second part of the grant takes advantage of the fact that yeast is the only eukaryote for which chromosomal origins of replication have been well characterized and can be isolated and studied in detail. We will investigate the protein/DNA and protein/protein interactions at ARS elements, yeast origins of replication. ARS binding factor I, ABFI, which we have shown to be involved in plasmid replication, the SSB, RP-A, and a new helicase we have identified that also binds to ARS DNA will be studied. If other critical initiation proteins are discovered, we will also add these to our work. We propose that one or more of the DNA binding proteins directs DNA polymerase to the origin of replication, and it is this proposed interaction that forms the link between the two parts of the proposal. Ultimately, we would like to reconstitute an unwinding and initiation event at an ARS with purified proteins. Study of the proteins described in this grant will provide useful background.