First Advisor

Justin Courcelle

Date of Publication

Summer 1-1-2012

Document Type

Thesis

Degree Name

Master of Science (M.S.) in Biology

Department

Biology

Language

English

Subjects

DNA repair, Escherichia coli, Ultraviolet radiation

DOI

10.15760/etd.767

Physical Description

1 online resource (vii, 71 p.) : ǂb ill. (some col.)

Abstract

Following low levels of UV exposure, Escherichia coli cells deficient in nucleotide excision repair recover and synthesize DNA at near wild type levels, an observation that formed the basis of the post replication recombination repair model. In this study, we characterized the DNA synthesis that occurs following UV-irradiation in the absence of nucleotide excision repair and show that although this synthesis resumes at near wild type levels, it is coincident with a high degree of cell death. We confirm that the replication occurring under these conditions involves extensive levels of strand exchange. However, cells undergoing this form of replication accumulate strand exchange intermediates that fail to resolve into discrete molecules, resulting in grossly filamentous, multinucleate cells. Taken together the results demonstrate that the DNA synthesis that occurs in UV-irradiated nucleotide excision repair mutants is aberrant and suggests that post replication repair is not an efficient mechanism to promote survival in the absence of nucleotide excision repair. The role that nucleotide excision repair plays in the recovery of replication following UV-induced DNA damage was further characterized by examining the specific role of UvrD in processing and restoring UV-arrested replication forks. UvrD is a helicase with functions associated with nucleotide excision repair and replication. UvrD catalyzes the removal of the damaged region by nucleotide excision repair proteins and removes the stretch of DNA incised during methyl-directed mismatch repair during replication. Recent biochemical studies have led to the proposal that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occurs normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

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Persistent Identifier

http://archives.pdx.edu/ds/psu/8501

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