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.
Recommended Citation
Church, Jonathan Spence, "A study of the effects of inosine incorporation into DNA due to defects in purine biosynthesis in Escherichia coli" (2012). Legacy Theses & Dissertations (2009 - 2024). 611.
https://scholarsarchive.library.albany.edu/legacy-etd/611