MECHANISM OF HUMAN DNA POLYMERASE DELTA, RF-C AND PCNA

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The human replication apparatus consists of over a dozen different proteins. Among them is PCNA, a trimer in the shape of a ring that completely encircles DNA. The PCNA ring slides freely along DNA and its function is to bind the DNA polymerase thereby holding it to DNA for long chain DNA synthesis, a behavior one might expect of a polymerase that needs to duplicate long chromosomes. The "PCNA clamp" does not assemble onto DNA by itself, it requires the matchmaking activity of the RFC complex. RFC is a 5 subunit complex that recognizes a primed DNA site and PCNA, and couples ATP hydrolysis to assemble PCNA around the DNA. Afterward, the DNA polymerase delta (and DNA polymerase epsilon) can assemble with the PCNA ring for highly processive synthesis of DNA. This proposal is an intensive investigation into the mechanism underlying the assembly of PCNA on DNA, and its behavior with DNA polymerase delta. How the PCNA ring is assembled around DNA by RFC and the role(s) of ATP in this process will be determined. Also, how the RFC complex coordinates its action on PCNA with the action of Pol delta on the same PCNA ring will be examined. During discontinuous synthesis on one strand of the chromosome, the DNA polymerase must be capable of coming on and off DNA for extension of numerous lagging strand fragments. If this polymerase is tied to DNA by the PCNA clamp, how does it recycle? Mechanisms for recycling of both the polymerase and the PCNA ring will be studied. These replication proteins are also involved in the repair of DNA and in regulation of the cell cycle.. Hence, a deeper understanding of the molecular mechanisms underlying the function of these proteins should also advance the fields of repair and cell cycle control.