FUNCTIONAL RELATIONSHIPS BETWEEN RHO, SSB, REP AND RECA PROTEINS IN ESCHERICHIA COLI: NOVEL ROLES FOR RHO AND RECA IN REPLICATION
Abstract
Escherichia coli transcription-termination factor Rho is an essential protein and mutations in the rho gene affect many cellular processes. However, the basis of Rho essentiality and of many of the defects conferred upon a cell when Rho is defective are not understood. In this thesis the UV sensitivity of rho mutants was studied to uncover roles for Rho other than transcription termination that might better account for the complex rho phenotype. A possible role for Rho in repair of UV-damaged DNA was investigated by examining the relative efficiency of repair pathways in rho mutants. Excision repair, error-prone repair, recA activation and recA amplification were all found to be normal in rho mutants. Polarity was suppressed by UV-irradiation (UV) of rho('+) cells in a manner analogous to the suppression characteristic of rho mutations. A hypothesis has been formulated which could explain the suppression of polarity by UV as well as the killing by UV of both rho('-) and wild-type cells. This hypothesis postulates that Rho protein is depleted after UV, and predicts that rho('+) strains with elevated Rho activity will be more UV-resistant than wild-type. In this work cya and crp mutants, known to have elevated Rho activity, were, in fact, found to be super-resistant to UV. Similarities in the UV-sensitive phenotypes of rep, ssb, and rho mutations suggested a possible functional relationship among Rep, SSB and Rho and this prompted the construction of the double mutants rho-ssb and rho-rep. These combinations turned out to extremely deleterious and suggested a role for Rho in replication. Furthermore, it was found that plasmids isolated from rho('-) cells were less supercoiled than when isolated from rho('+) cells, which suggested a role for Rho in determining DNA structure, possibly as a topoisomerase or helicase. rep-(DELTA)recA double mutants were found to be conditionally lethal and defective in transposon and plasmid maintenance. One rep-(DELTA)recA combination caused a conditional block in DNA synthesis, suggesting a previously unrecognized role for RecA in the unwinding of DNA during replication.
Degree
Ph.D.
Subject Area
Biology
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