The investigator will study genetic recombination using assay systems that monitor recombination between repeated sequences in the bacterial chromosome. Unlike crosses, these assays provide no DNA ends; spontaneous DNA damage initiates the exchange. In a duplication segregation assay, the main source of initiating structures appears to be damage to DNA caused by reactive oxygen species. The metabolic steps involved in generating reactive oxygen species will be identified. Oxidative DNA damage is thought to play a major role in carcinogenesis and aging. Chromosome inversions can form by recombination between separated inverse order repeats. For particular chromosome regions, inversion is impossible in a wild type strain but is allowed by a mutation in the tus gene, whose product terminates replication at Ter sites. It is proposed that non-permissive replication must cross an active Ter site; such replication is possible only in the absence of Tus protein. An assay for reciprocality of recombination suggests that 25% of the exchanges which form a duplication are reciprocal; they also form the corresponding deletion. This apparent reciprocality may be due to serial half reciprocal exchanges which occur by initiation of replication forks in the course of recombination. These studies provide an experimental approach for the role of replication in recombination. A model will be tested which explains the Cairns phenomenon of apparent adaptive mutability without requiring any increase in intrinsic mutation rate. The model involves selective amplification of the mutant allele and includes (but does not rely on) a hypothesized new mutation type (the do-loop) by which short DNA sequences can by highly amplified by rolling circle replication initiated by the repair process. This model offers a way of explaining the genetic basis of triplet expansion diseases and the evolution of new genes. This proposed work investigates illegitimate double strain break repair, a process by which sequences can transpose into spontaneous breaks without involvement of any transposable element or transposase. This process may be a major cause of spontaneous chromosome rearrangements. In addition, chromosome position effects on recombination, transposition and mutation will be addressed; preliminary observations suggest that chromosome context can have a big effect on all of these processes. Finally, the proposal includes studying the behavior of the unusual transposable element IS200, which is found in virtually all Salmonella strains, but transposes very rarely.
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