SPONTANEOUS AND GENOTOXICANT INDUCED MUTATION MECHANISMS

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The long term goal of this research is to understand the molecular mechanisms involved in the spontaneous and genotoxicant induced mutagenesis of DNA sequences that have the potential to adopt alternate secondary structures. Spontaneous mutation and mutations induced by environmental carcinogens can lead to genetic disease and cancer in humans. In this proposal we will use E. coli to test the hypothesis that certain sequences of DNA can form secondary structure substrates which promote specific mutation events at a high frequency. We will then test whether environmental mutagens enhance the frequency of these events. In a separate study we will apply this system (once it is clearly understood) to a whole animal system. Secondary structure substrates for mutagenesis can include cruciform structures, formed from inverted repeated DNA sequences; Z-DNA, formed in regions of alternating purine/pyrimidine sequence; and slipped mis-paired structures, formed during DNA replication in direct repeated sequences. DNA that can adopt such alternate conformations occurs widely in human genomes. We have developed sensitive in vivo assays for cruciforms and Z-DNA and plan to determine the relationship between the in vivo existence of alternate conformational forms of DNA and their relative mutagenic potential. We can test various models for sequence directed deletion using our numerous series of inverted repeats that can form cruciforms in vivo. These models involve direct loss (possibly excision) of cruciform arms, 'resolution" of cruciforms (as intermediates in genetic recombination), and slipped mispairing during DNA replication. For example, we will correlate the fraction of inverted repeats existing as cruciforms in vivo with the genetic instability of the inverted repeat. From such analysis we can determine if the formation of cruciforms itself leads to mutation or if mutation is the result of a stable hairpin arm formed during replication. We have also developed an assay for the frequency of deletion and duplication between direct repeats of 17-26 bp DNA sequences that can form defined secondary structure substrates. Using this system we should be able to identify the molecular intermediates and mechanisms involved in deletion and duplication between direct repeats. We will also investigate the role of genes involved in, recombination, DNA repair, and replication in spontaneous DNA directed mutagenesis. The effect of environmental mutagens on the frequency of DNA directed mutation should not only serve to identify agents that facilitate this type of event, but should also provide insight into mechanisms.