Gcn4p does not appear to be necessary for initiation of recombination, unlike the other three proteins, which are required for recombination. The Bas1p and Bas2p binding sites can be replaced with extra Rap1p binding sites. Increasing the number of Rap1p binding sites increases the level of meiotic recombination, but does not alter the level of mitotic recombination, indicating that Rap1's function in recombination is meiosis-specific.
The Rap1 DNA-binding protein has been implicated in many different cellular processes, including activation of transcription, maintenance of silencing at the mating type locus, and regulation of telomere length in addition to its role in recombination. Click here to see a diagram of the Rap1p domains. I have been attempting to delineate the region of Rap1p responsible for meiotic recombination initiation. Essentially, we believe that only the activation domain and the DNA binding domain of the protein are required to initiate meiotic recombination. This result explains why recombination and transcription have been intimately linked.
In addition to transcription, the state of the chromatin in the HIS4 promoter region correlates with the initiation of recombination. A previous study by the lab demonstrated that the meiotic recombination hotspot was in a region of open chromatin, as demonstrated by DNaseI hypersensitive areas. In collaboration with members of the Griffith lab at UNC, I have investigated the effect on meiotic recombination, transcription, and chromatin state of a 5 basepair DNA sequence that previously was shown to exclude nucleosomes in vitro. This sequence was repeated tandemly either 12 or 48 times. These two tracts were used to replace the HIS4 hotspot. The chromatin state at HIS4 was examined with the longer tract; a DNaseI hypersensitive area comparable to the wildtype sequence region was detected. Both tracts activate transcription at HIS4. The shorter tract acts as a meiotic recombination hotspot; the longer tract acts as a coldspot, actively repressing recombination at a nearby introduced hotspot.
In addition to my work with recombination initiation, I also am working on the manner in which DNA mismatches that occur as a consequence of recombination are repaired during meiosis. One paper has appeared on this topic, and I am continuing to work on this project. I have shown that yeast possesses at least three pathways for the repair of mismatches that occur during heteroduplex formation. One is akin to the mitotic DNA mismatch repair pathway that functions during vegetative growth, and acts on small base-base mismatches and loops. Two more pathways are responsible for the correction of larger loops, and involve genes in both DNA mismatch repair and in nucleotide excision repair.
Another area of investigation is the nature of gene conversion tracts at HIS4. I showed that at HIS4, the polarity gradient for recombination is mostly likely due to a transition from gene conversion repair to restoration repair. This study also demonstrated the existence of restoration repair during recombination; a type of repair that had not been proven in yeast prior to this because the repair event recreates the normal Mendelian segregation seen if no recombination had taken place. This work recently appeared in Genetics. A number of other projects are being pursued. Check back later for more details!
All of this work is conducted in the lab of Tom Petes, where I am a postdoctoral fellow, having received my Ph.D from MIT a few years back. I am currently being funded as a Special Fellow of the Leukemia Society of America.
Here's a copy of my CV.
