Date of Award

1-1-2012

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (vi, 47 pages) : PDF file, illustrations (some color)

Dissertation/Thesis Chair

Richard Cunningham

Committee Members

Thomas Begley, Robert Osuna

Keywords

inosine, purine biosynthesis, Purines, Inosine, DNA damage

Subject Categories

Molecular Biology

Abstract

Deamination of purine bases can result in the formation of xanthine and hypoxanthine which can be miscoding and mutagenic in DNA. There are several mechanisms for the introduction of deaminated bases into DNA including simple hydrolysis, nitrosative chemistry and through the action of deaminase enzymes. A fourth method was recently presented which describes how deaminated purines can be incorporated into DNA due to defects in purine biosynthesis. Using fluctuation analysis, spontaneous mutation rates were studied in bacterial mutants that were deficient in specific genes involved in purine biosynthesis and dNTP precursor pool maintenance, including purA (adenylosuccinate synthetase), guaA (GMP synthetase), guaB (IMP dehydrogenase), rdgB (dITP pyrophosphatase) and nfi (endonuclease V). Synergistic increases in mutation rate were observed as combinations of gene deletions were introduced that would result in increased levels of dITP in nucleotide precursor pools. SOS induction levels were measured in these strains using Western blot analysis of RecA protein levels. This assay was performed in order to rule out SOS induced mutagenesis as being the root cause of the increases in observed mutation rate. RecA expression levels increased as deletions to purine biosynthesis genes were introduced. A 9-fold increase in RecA expression levels was observed in the purA guaB rdgB triple mutant. Expression levels returned to wild type levels with the deletion of the nfi gene while mutation rates remained high implying that SOS induction is not the main cause of the increased mutation rates we observed. These observations provide evidence that, as inosine is incorporated into DNA due to deficiencies in purine metabolism, mutation rates increase and the SOS response is induced when endonuclease V is active. A possible method for limiting incorporation of and repairing already incorporated deaminated purines is also described.

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