REGULATION OF THE S POMBE M26 RECOMBINATION HOTSPOT

S. pombe的ade6-M26突变先前已被确定为减数分裂重组热点。热点是由ade6基因5'端附近的单个G产T翻转引起的。该突变产生了一个序列，5-ATGACGT-3'，它对热点活动至关重要，并且在基因组的其他位置也有活性。M26热点活性需要结合由atf1和pcr1基因编码的异二聚体转录因子，但热点活性的调控机制尚不清楚。Aft1和Pcr1存在于有丝分裂和减数分裂过程中，而M26仅在减数分裂过程中活跃。然而，最近Atf1被鉴定为一种磷酸化蛋白，并将进行实验来确定磷酸化在调节热点活性中的作用。磷酸化在体外与M26结合中的作用将通过使用放射性标记的M26探针进行凝胶阻滞测定。使用atf1::HA6H标记的等位基因和IP-PCR方法，还将测量与M26的体内结合。为了确定调节M26热点的其他潜在因素，将进行有丝分裂激活M26的突变筛选。高重组基因表型可以很容易地识别通过集落乳头实验。因此，这种方法的一个巨大优势是可以相当迅速地筛选和识别大量突变。通过这种筛选确定的基因将提供对同源重组机制的见解，这将增加我们对与该过程相关的人类疾病的理解，包括许多类型的癌症。

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.