The bacteriophage PRD1 is a lipid bearing phage that infects a wide variety of gram-negative bacteria, including Escherichia coli and Salmonella typhimurium when they harbor the appropriate plasmid. It contains a linear duplex DNA molecule that is covalently bound by its 5' ends to a terminal protein. Like adenovirus and the Bacillus phage φ29, PRD1 specifies its own DNA polymerase which is able to utilize the phage encoded terminal protein to prime DNA synthesis. In addition to these two proteins, PRD1 also specifies an additional replication protein (p12) of unknown function. We have sequenced the origins of replication (termini of the genome) as well as the right most 1700 bp of the bacteriophage PRD1 genome. The right most 1700 bp encompasses the right early region and completes the sequence of all PRD1 early functions. We report here that the PRD1 genome contains a perfect 111 bp inverted terminal repeat. Furthermore, statistical analyses of the right 1700 bp, as well as the examination of transcription and translation signals has allowed us to assign gene XII to an open reading frame and to infer the direction of both early and late transcription. Gene XII, which has been implicated in the replication process and the regulation of gene expression is predicted to encode a 16.7 Kdal protein. Data base searches have revealed a possible evolutionary relationship between this protein and the ε-subunit of E. coli DNA polymerase III. We have also mapped right, early transcription of the PRD1 genome. This has corroborated our inference concerning the direction of right early transcription and confirmed our assignment of gene XII to an open reading frame. It has also revealed that two putative rho-independent terminators are functional in vitro and that the putative right early promoter is utilized in vivo and in vitro. The data presented here have permitted us to ascertain the general genomic and transcriptional organization of PRD1 and to predict the primary structure of the product of gene XII. These results, in turn, have allowed us to develop hypotheses concerning the evolution of linear, protein primed DNA's and the function of gene XII.

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DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.

Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 101-120). Also available via World Wide Web.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 179 p.; also includes graphics. Includes bibliographical references (p. 155-179). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 1997. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2005. / "A dissertation in pharmaceutical sciences and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed June 23, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 327-353). Online version of the print edition.