The overall goal of this project is to define, in enzymatic and molecular terms, the mechanism by which a duplex DNA molecule is replicated. Bacteriophage T7, whose duplex chromosome encodes the majority of the proteins responsible for its replication, will be used as a model system. The initiation of replication at the primary origin, whose location and nucleotide sequence are known, will be studied by (1) establishing the role of each of the genetic elements that constitute the primary origin by using in vitro mutagenesis, (ii) reconstituting the initiation event using purified proteins, (iii) determining the role of T7 RNA polymerase in initiation, (iv) identifying the sites of transition from RNA to DNA synthesis both in vivo and in vitro, and (v) identifying the factors necessary to achieve bidirectional replication from the origin. Secondary origins of replication will be mapped in order to compare initiation mechanisms. Major emphasis will be placed on the further characterization of the replication proteins and their interaction with one another and with the DNA template. In order to have available sufficient quantities of the replication proteins and to carry out in vitro mutagenesis, the replication genes will be cloned and overexpressed. The basis for the existence of the two forms of T7 DNa polymerase will be pursued, as well as the role of its subunit, E. coli thioredoxin, in replication. The mechanism of action of the multifunctional gene 4 protein will be studied with regard to its helicase, primase, and translocation activities. Two novel proteins, the E. coli optA protein and the T7 gene 1.2 protein, whose expression is regulated post-transcriptionally, will be purified and characterized. The physical interaction of proteins such as T7 DNA polymerase and gene 4 protein will be documented and the complex isolated and characterized. Using the purified proteins, a functional replication fork will be reconstituted using a recently constructed, preformed, topologically stable replication fork. The preformed replication fork will be used to examine such parameters as the microscopic rate of fork movement and the coupling of leading and lagging strand synthesis. The metabolism of concatemers will be studied, including the roles of the gene 2 protein (inhibitor of E. coli RNA polymerase) and the gene 18 and 19 proteins (processing of concatemers).
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