Advisor

Coats, Karen

Committee Member

Downer, Donald

Committee Member

Wise, Dwayne

Committee Member

Boyle, John

Committee Member

Gavini, Nara

Date of Degree

5-1-2007

Document Type

Dissertation - Open Access

Major

Biological Sciences

Degree Name

Doctor of Philosophy

College

College of Arts and Sciences

Department

Department of Biological Sciences

Abstract

Accurate replication of the genome is essential for reproduction in all cells. However, even under normal conditions, the replication machinery may face a variety of impediments that can prevent it from completing its task. The mechanism by which cells overcome these hurdles is likely to vary depending upon the nature of the obstacle. Both UV irradiation and inactivation of replicative proteins in DnaB can inhibit the progression of the DNA replication machinery. However, the mechanism by which replication recovers following UV irradiation is different from the mechanism of recovery following the inactivation of the replicative proteins. Previous results show that following UVinduced damage in Escherichia coli, the replication fork is maintained and protected from extensive degradation by RecF, RecO, and RecR until replication can resume. By contrast, replication does not recover following inactivation of the replication protein DnaB, and the nascent DNA is extensively degraded irrespective of whether RecF is present. In this study, we verified DNA replication arrest by monitoring the total DNA accumulation and rate of DNA synthesis following UV-induced DNA damage and inactivation of thermosensitive replication alleles, such as dnaB266. We measured the amount of nascent DNA degradation, allowing us to determine how the newly synthesized strand of DNA is affected following replication fork arrest. Our data indicate that following inactivation of DnaB266, the replication fork is not maintained and is subject to extensive degradation. The degradation that occurs after DnaB266 inactivation is partially reduced in the absence of RecF-O-R, RecJ, and ExoI, suggesting that DNA processing by these enzymes occurs after DnaB arrest. In addition, two-dimensional agarose gel analysis revealed that unique structural intermediates accumulated following inactivation of DnaB266. These observations indicate that the recovery of replication when impeded by DNA lesions, such as those produced by UVirradiation, is maintained and processed through mechanisms that do not resemble the events occurring when replication proteins are inactivated.

URI

https://hdl.handle.net/11668/16198

Comments

dnaB||temperature sensitivity||dnaE||DNA replication--Effect of radiation on.||DNA damage.||DNA repair.||Escherichia coli--Effect of ultraviolet radiation on.||Escherichia coli--Genetics--Effect of radiation on.||Microbial enzymes.

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