This is a continuation of a long running genetic investigation on the mechanism of recombination in yeast. The experimental effort is focused on understanding the molecular events involved in initiating meiotic recombination. In particular there will be an investigation of the change in DNA structure that appears to be a prerequisite for initiation of recombination. A well characterized recombination hotspot upstream of the HIS4 gene will be investigated. Recent studies have established that recombination at the hotspot is associated with formation of a transient double strand break. Petes plans to study the nature of the break so as to determine the structure at the nucleotide level. Information obtained will provide insight into the specificity of the nuclease responsible which will ultimately be useful in a planned effort to identify this important nuclease. Identification of this nuclease will be pursued by both genetic and biochemical means. The genetic approach will rely on the lethality that occurs when double strand breaks are introduced into rad52 strains. A screen will be implemented in which a library under control of the galactose inducible GAL1 promoter will be introduced into a rad52 mutant. Transformants will be screened for sensitive clones when plated on galactose medium. The rationale for the screen is that lethality on galactose implies induction of an endonuclease. A second approach will be a reverse genetics approach that will follow from biochemical isolation of the endonuclease. Extracts from cells committed to meiosis will be assayed for endonuclease activity and then fractionated so as to purify the activity. This effort will be carried out in collaboration with Dr. Steven Matson an accomplished DNA biochemist. Once the activity is purified the gene will be obtained by standard reverse genetic methods, a null mutant will be constructed, and the recombination phenotype characterized. The chromatin structure of the HIS4 hotspot will also be examined. The double strand break site will be mapped and the position compared to DNaseI hypersensitive sites. The interesting finding would be that the positions of the cut sites are identical, giving strong indication that nuclease accessible regions of the chromatin are the sites of initiation of recombination. Another area of investigation will focus on the role of specific proteins in forming the HIS4 hotspot. Two classes of proteins will be investigated. These include proteins that bind to specific sequences in the HIS4 upstream region and products of genes that globally control chromatin structure. Of the former class the activator/repressor Rap1 has already been shown to exert an effect on HIS4 recombination so it, in particular will be studied in more detail. This will be done by examining the effect of a variety of rap1 mutants on recombination. Mutants in histone and HMG genes which have a general effect of chromatin structure will also be examined. Processing of the DNA ends after introduction of DSBs will also be examined. The goal here is to learn the rules for how DSBs are repaired and to set up a system for a nick-directed mismatch repair system.
A final aim i nvolves investigating the introduction of DSBs in a recombination hotspot in the mouse MHC locus.
Showing the most recent 10 out of 102 publications
