The in vivo roles of the RdgB enzyme in Escherichia coli and the detection of rare non-canonical DNA precursors.

摘要

Adenine, guanine, cytosine, and thymine deoxyribonucleotides make up the pool of canonical DNA precursors in living organisms. The incorporation of non-canonical DNA precursors into DNA during replication can lead to mutagenesis, chromosomal damage, or both. The approach taken by living organisms to address this problem is to have a set of enzymes that sanitize the DNA precursor pools, ensuring a balanced, high-quality source of canonical DNA precursors for replication. The RdgB enzyme of Escherichia coli hydrolyzes pyrophosphate from 2'-deoxyinosine triphosphate (dITP), preventing the incorporation of this non-canonical DNA precursor into DNA during replication. The rdgB mutant of E. coli suffers DNA double-strand breaks and is a synthetic lethal mutation in combination with either the recA or recBC mutations, indicating a dependence on recombinational DNA repair. This dissertation describes the consequences of dITP incorporation into DNA in regards to mutagenesis and chromosomal damage. While the incorporation of deoxyinosine into DNA via dITP is known to lead to DNA double strand breaks, I found that dITP incorporation does not increase mutagenesis. dITP is thought to arise endogenously via spurious metabolic reactions. By isolating suppressors of the rdgB recA synthetic lethality, I have identified several enzymes involved in purine metabolism which appear to contribute to the contamination of the DNA precursor pools with dITP. I also measured the type and extent of DNA precursor pool contamination in rdgB mutants by developing a powerful method for large-scale purification and analysis of cellular nucleotide pools; using this technique, I directly measured the concentration of dITP at micromolar intracellular concentrations in an rdgB-deficient strain of E. coli. This new method was also applicable to studying gross perturbations of DNA precursor pools, in addition to detecting contamination of DNA precursor pools with rare non-canonical nucleotides. I applied this sensitive method to study the DNA precursor pool perturbations that occur during thymine starvation in a thymine auxotroph of E. coli.