STARVATION, MUTATION RATES, AND THE STRINGENT RESPONSE

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It is estimated that DNA damage ultimately causes 80 to 90 percent of all human cancers. The long-term objective of this research is to understand the relationships between mutation rates, DNA repair, transcription, starvation, and the accumulation of guanosine tetraphosphate (ppGpp). The model system under investigation is E. coli K12, and the mutation rates analyzed are reversions of amino acid auxotrophs to prototrophy. The reversion rates of two isogenic E. coli K12 auxotrophs differing only in rel A have been determined in the absence or presence of serine hydroxamate, which provokes the stringent response. Reversion rates of leu B- and arg H- are both significantly higher in the rel A+ compared to the rel A- strain, and the reversion rate of leu B- in both strains is enhanced by the presence of serine hydroxamate. A positive correlation has also been established between reversion rates and the accumulation of ppGpp in the absence and presence of serine hydroxamate. Specific objectives for future investigations are: 1. To determine the dependance of reversion rates and the accumulation of ppGpp in the isogenic strains on: (a) ppGpp degradation, controlled by ppGpp 3' pyrophosphohydrolase which is encoded by the spo T gene; (b) Homologous recombination functions which are dependant upon the rec ABC gene products; (c) SOS mutator activity, controlled by the rec A, lex A, and umu DC genes. (d) The transcription-repair coupling factor, encoded by the mfd gene. 2. To determine the generality and the specificity of our results with the isogenic auxotrophs by: (a)Introducing a mutant gene known to undergo post-plating reversions ("adaptive mutations") into the isogenic strains, in order to measure both growth and post-plating reversion rates in the rel A+ and rel A-strains; (b) Measuring reversion rates and ppGpp accumulation of the ara-, xyl- or gal- mutant genes, which are markers that are also present in the isogenic rel A+ and rel A- strains, during starvation for amino acids or carbohydrates. Measure leu B- reversion rates in both strains during carbohydrate starvation. (c) Measure mutation rates of a gene the transcription of which is not stimulated by the stringent response. 3. To determine the sequence the leu B- gene in the rel A+ and rel A- strains and in revertants of these strains and their transductants.