Spontaneous mutation and mutations induced by environmental carcinogens can lead to human genetic disease and cancer. In this proposal, the applicant will use E. coli to continue to test the hypothesis that certain sequences of DNA can form secondary structure substrates, which promote specific mutation events at a high frequency or, in some cases, prevent a specific mutation. Secondary structure substrates for mutagenesis can include cruciform structures, formed from inverted repeated DNA sequences; slipped mis-paired structures, formed during DNA replication in direct repeated sequences; and other non Watson-Crick duplex, triplex, or quadruplex structures. DNA that can adopt such alternative conformations occurs widely in the human genome. Dr. Sinden's laboratory has developed a sensitive in vivo assay for cruciforms and will continue to determine the relationship between the in vivo existence of cruciforms and their relative mutagenic potential. Inverted repeats appear to be deleted by misalignment stabilized by a hairpin formed as a cruciform or in single-stranded DNA in the cell. His lab has also developed a chloramphenicol resistance reversion assay to measure the frequency of deletion and duplication between direct repeats of 17-23 bp """"""""mutation insert"""""""" DNA sequences that can form defined secondary structure substrates. Using this system, they will continue to identify the molecular intermediates and mechanisms involved in deletion and duplication between direct repeats. Dr. Sinden's laboratory will continue to test out the hypothesis that misalignment stabilized by a three-way junction leading to deletion occurs preferentially in the lagging strand. By the synthesis and mutational analysis of specifically designed quasipalindromes, they will continue to investigate the molecular mechanism responsible for the correction of quasipalindromes to perfect inverted repeats, which appears to involve an intermolecular strand switch that happens preferentially in the leading strand during DNA replication. They will also investigate the role of genes involved in DNA replication, recombination, and repair in spontaneous DNA directed mutagenesis to identify proteins and enzymes responsible for the introduction of mutations into the genome. The influence 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 molecular mechanisms.
