The long term goal of this project is to gain a better understanding of the mechanism of DNA replication by reconstruction in vitro of events that take place at the replication fork. Our recent progress toward this goal has included: preparation of intact, homogeneous DNA polymerase alpha/primase; purification to homogeneity of a new accessory factor for DNA polymerase alpha/primase (alpha accessory factor or AAF) and characterization of its properties and novel mechanism of action; development of a system that emulates discontinuous DNA synthesis on the lagging strand using five purified mouse proteins; identification of a RNA primer removal mechanism; and purification and characterization of two DNA polymerases delta from mouse cells, one of which is the catalytic subunit of the other. It is proposed that this work be continued as follows: 1) Carry out additional studies on several of the proteins thus far purified; particularly, AAF, the 5'-exonuclease, DNA polymerase alpha/primase, and RNase H-1. This would include scale-up of the purification procedure to make available larger amounts of protein; more detailed studies on physical and catalytic properties and mechanism of action; preparation of antibodies as a reagent for polypeptide identification and specific inhibition of activity; and selection of cDNA as a source (through overexpression) of the protein and as another route to information on structure and function. A special effort will be made to clarify the roles of putative replication proteins, e.g. AAF, the 5'- exonuclease and RNase H-1. 2) Purify and characterize additional replication proteins by specific assays; this includes helicases and additional RNA primer-removing activities. 3) Develop an in vitro DNA replication system dependent on Epstein-Barr virus (EBV) oriP and EBNA-1, to study its components and mechanism. 4) Construct a preformed replication fork on which to synthesize leading and lagging strands from nine purified proteins; this, as well as the EBV system, will be used to detect, purify and characterize additional proteins that participate in DNA replication. It is believed that greater knowledge of the mechanism of DNA replication will result in health benefits because of its central importance in problems such as errors in replication and injury/repair of DNA and their relationship to mutations and carcinogenesis, as well as providing a basis for the rational design and use of chemotherapeutic agents. In particular, it is hoped that an understanding of the structure and mechanism of EBV oriP and EBNA-1 and the cellular proteins with which they interact will contribute to knowledge concerning the pathogenesis of EBV-related tumors and ultimately help in their control.
