The ade6-M26 mutation of S. pombe has been previously identified as a meiotic recombination hotspot. The hotspot results from a single G yields T transversion near the 5' end of the ade6 gene. The mutation creates a sequence, 5-ATGACGT-3', which is essential for hotspot activity and is active at other locations in the genome. M26 hotspot activity requires binding of a heterodimeric transcription factor encoded by the atf1 and pcr1 genes, but the mechanism by which hotspot activity is regulated is not known. Aft1 and Pcr1 are present throughout mitosis and meiosis, but M26 is only active during meiosis. Recently, however, Atf1 was identified as a phosphoprotein, and experiments will be performed to determine the role of phosphorylation in regulating hotspot activity. The role of phosphorylation in binding to M26 in vitro will be performed by gel retardation assays using a radiolabelled M26 probe. In vivo binding to M26 will also be measured by use of an atf1::HA6H tagged allele, and the method of IP-PCR. In order to identify other potential factors regulating the M26 hotspot, a screen for mutations that activate M26 mitotically will be performed. The hyper-recombinogenic phenotype can be readily identified by a colony papillation assay. Therefore, a tremendous advantage of this approach is that large numbers of mutations can be screened and identified quite rapidly. Genes identified by this screen will provide insights into the mechanisms of homologous recombination, which will increase our understanding of human diseases associated with this process, including many types of cancer.
|Steiner, Walter W; Schreckhise, Randall W; Smith, Gerald R (2002) Meiotic DNA breaks at the S. pombe recombination hot spot M26. Mol Cell 9:847-55|