Date of Award


Degree Type


Degree Name

Doctor of Philosophy


My work has involved the sequencing of the Escherichia coli rep gene. This gene codes for the Rep helicase, a ssDNA-dependent ATPase required by some phages ({dollar}\Phi{dollar}X174, fd, P2) for their replication.;The section of E. coli chromosome known to contain the rep gene, which I have sequenced, has only one transcribed open reading frame of the correct size to encode the Rep protein. The predicted N-terminal amino acids and the predicted total amino acid composition are in agreement with those determined from purified Rep protein.;The proposed amino acid sequence of Rep contains in its N terminus a peptide common to all ATP-binding enzymes: G/A-X{dollar}\sb4{dollar}-GKT-X{dollar}\sb6{dollar}-I. It is also possible to use the primary amino acid sequence data to predict a secondary structure for the Rep protein. The amino acid sequence of the Rep protein was compared with others already present in the protein sequence data bank; DNA helicase II the uvrD gene product, was identified as the only protein with significant homology with Rep.;In order to correlate structural features with functional protein domains two approaches were used. The first approach involved creating minor deletions in the gene and assaying for some of Rep's activities.;The second approach has been to investigate the dasC mutation (das stands for dnaA suppressor). This mutation had been mapped to the vicinity of the rep gene. The dasC phenotype (growth at 42{dollar}\sp\circ{dollar}C of an E. coli strain carrying the dnaA46 temperature sensitive allele) appears to be due to a lesion in the DNA downstream from the rep gene "dasC" which represses the ability of Rep to complement the dnaA46 mutant. The rep alleles isolated from dasC and wild type strains confer the temperature-resistant phenotype.



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