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).

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37AI006045-23
Application #
3480399
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1976-01-01
Project End
1990-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
23
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Mendelman, L V; Beauchamp, B B; Richardson, C C (1994) Requirement for a zinc motif for template recognition by the bacteriophage T7 primase. EMBO J 13:3909-16
Kim, Y T; Richardson, C C (1994) Acidic carboxyl-terminal domain of gene 2.5 protein of bacteriophage T7 is essential for protein-protein interactions. J Biol Chem 269:5270-8
Hine, A V; Richardson, C C (1994) A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7. Proc Natl Acad Sci U S A 91:12327-31
Debyser, Z; Tabor, S; Richardson, C C (1994) Coordination of leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7. Cell 77:157-66
Liu, Q; Richardson, C C (1993) Gene 5.5 protein of bacteriophage T7 inhibits the nucleoid protein H-NS of Escherichia coli. Proc Natl Acad Sci U S A 90:1761-5
Kim, Y T; Richardson, C C (1993) Bacteriophage T7 gene 2.5 protein: an essential protein for DNA replication. Proc Natl Acad Sci U S A 90:10173-7
Mendelman, L V; Notarnicola, S M; Richardson, C C (1993) Evidence for distinct primase and helicase domains in the 63-kDa gene 4 protein of bacteriophage T7. Characterization of nucleotide binding site mutant. J Biol Chem 268:27208-13
Wurgler, S M; Richardson, C C (1993) DNA binding properties of the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli. J Biol Chem 268:20046-54
Notarnicola, S M; Richardson, C C (1993) The nucleotide binding site of the helicase/primase of bacteriophage T7. Interaction of mutant and wild-type proteins. J Biol Chem 268:27198-207
Kim, Y T; Tabor, S; Churchich, J E et al. (1992) Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7. J Biol Chem 267:15032-40

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