Research Summary Nucleic Acids Enzymology Genetic recombination is the intracellular process that moves DNA sequence information from one genomic location to another. The molecular mechanisms that regulate recombination- i.e., the mechanisms that regulate where and when it occurs- operate during the first step in the reaction, which for eukaryotic recombination is the formation of a double-stranded break (DSB) in DNA. To increase our understanding of these regulatory mechanisms, our laboratory investigates the formation and processing of DSBs in the yeast Saccharomyces cerevisiae. Mating Type Switching Much of our attention focuses on the recombination reaction that switches mating types in yeast. Haploid yeast cells occur in two mating types designated a and a. These types are determined by a master regulatory locus on Chromosome III known as MAT, which contains the MATa allele in a cells and the MATa allele in a cells. Chromosome III also carries two other loci involved in mating type determination called HMR and HML, which contain unexpressed or silent copies of MATa and MATa, respectively. Once per cell division, the allele at the MAT locus is replaced by the alternative MAT allele from one of the silent loci. This interconversion, which produces a phenotypic switch in mating type, is mediated by a genetic recombination reaction that shares many similarities with "standard" mitotic and meiotic recombination. For instance, like other eukaryotic recombination reactions, mating type switching initiates with a DSB. Specifically, it is initiated when an endogenous, site-specific endonuclease known as HO generates a DSB at a unique site in the MAT locus. Interestingly, however, HO does not cleave identical sequences at the two silent loci. We are using this example of spatial regulation as a model system to learn how cells determine where to initiate DSB formation. In the mating type system, it was found that the HO recognition sequence at MAT is hypersensitive to DNase I digestion, whereas the same sequence at HML or HMR is resistant. This suggested to us that a protein(s) required for HO cleavage may be present in vivo at the HO recognition site of MAT, but absent from the same sequences in the silent loci. To test this model, we developed a band shift assay to determine whether any non-HO protein(s) binds to double-stranded oligonucleotides containing the HO recognition site and flanking sequences. This assay showed that yeast extracts contain a small dimeric protein, which we named YZBP, that binds as predicted. Two DNA sequences immediately flanking the HO recognition sequence are required for the band shift. One of these is the binding site for YZBP. Preliminary results indicate that the other site may bind Pho2, a general transcription factor that regulates the expression of many yeast genes. The involvement of such a protein in DSB formation may explain why DSBs are normally targeted to gene promoters during meiotic recombination. We hypothesize that YZBP and, possibly, Pho2 act as cofactors with HO to form the DSB at MAT. We have purified HO endonuclease to homogeneity and shown that the enzyme binds to DNA with the same affinity as a typical bacterial restriction endonuclease, but cleaves approximately 500-fold less efficiently, suggesting that HO may be unable to act in isolation. This observation, coupled with the fact that the YZBP and putative Pho2 binding sites are required for DSB formation in vivo, supports our model that HO, YZBP, and Pho2 cooperate to form the DSB that initiates mating type switching. Identification of Genes Regulated in Response to the Chemical Induction of DSBs The preceding project analyzes the molecular mechanisms by which a single, well-characterized DSB is formed in vivo. Recently, we have started a new project to determine what happens after DSB formation. This project has two specific goals: to identify new proteins involved in the repair of DSBs, and to identify regulatory proteins that control the cell cycle arrest known to occur following DSB formation. We are employing a new procedure, known as differential display, to identify genes that exhibit altered expression patterns in response to the chemical induction of DSBs. Although this project is still in its preliminary stages, we have already identified several genes that may regulate cell cycle progression and one gene whose translation product has a DNA-binding motif consistent with a role in recombinational repair. Publications Wang, R., Jin, Y., and Norris, D. (1997). Identification of a protein that binds to the Ho recognition sequence at the yeast mating type locus. Mol. Cell. Biol. 17: 770-777. Jin, Y., Binkowski, G., Simon, L. D., and Norris, D. (1997). Ho endonuclease cleaves MAT DNA in vitro by an inefficient stoichiometric reaction mechanism. J. Biol. Chem. 272: 7352-7359. Tsui, K., Simon, L., and Norris, D. (1997). Progression into the first meiotic division is sensitive to histone H2A-H2B dimer concentration in Saccharomyces cerevisiae. Genetics 145: 647-659. Lab Support Dr. Kenji Hashimoto, Visiting Scientist Dr. Vijayalakshimi Nagaraj, Postdoctoral Fellow Kochung Tsui, Graduate Fellow Rong Wang, Graduate Fellow Yongjie Jin, Graduate Student Zhiheng Xu, Graduate Assistant