The yeast Saccharomyces cerevisiae will be used as a model system to study two processes that influence the stability of the eukaryotic genome: recombination between repeated sequences and mutation. Large numbers of repeated sequences are found dispersed throughout eukaryotic genomes and, in principle, can serve as potential substrates for recombination events. While aspects of the sequences themselves, such as size and degree of sequence divergence, clearly influence recombination between diverged sequences.
The first aim of this proposal will be to define the role of sequence divergence as a direct impediment to the recombination machinery versus the role of the mismatch repair machinery in regulating recombination. An intron-based recombination system that allows the degree of sequence homology between substrates to be varied systematically will be used for this analysis, and both mitotic and meiotic recombination events will examined. In addition to sequence considerations, it must be appreciated that eukaryotic DNA is packaged with nucleosomes into chromatin, which is in turn highly compacted. The basic metabolic processes that must occur on DNA (recombination, replication, repair, and transcription) require a relaxation of the chromatin structure and it has been appreciated that one process may exploit the open chromatin structure associated with the occurrence of another process. In addition, the relaxation of chromosome compaction may also render the DNA more accessible to endogenous DNA damaging agents and hence may impact on the need for DNA repair processes. The second and third aims of this proposal will be to determine the basis for the stimulatory effects of high levels of transcription on the processes of recombination and mutation, respectively. Transcriptionally active chromatin may be more accessible to the recombination machinery and/or may suffer damage that is repaired by recombination. The relative contributions of these two factors will be examined by experimentally manipulating the transcription of donor versus recipient sequences in recombination events. In relation to the stimulation of mutation events by high levels of transcription, Transcriptionally active chromatin may be more accessible to endogenous DNA damaging agents, or high levels of transcription may influence DNA repair processes. A variety of genetic and molecular approaches will be used to examine the effects of transcription of DNA damage and repair in yeast.
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