Bleomycin is an anticancer drug that has important clinical applications and is of biological interest because of its unique ability to bind and cleave DNA. Tissues with low levels of this enzyme, particularly lung, are very sensitive to bleomycin and tumors with high levels of the enzyme become resistant to the drug. The only known function of BH is to hydrolyze bleomycin. Even though most organisms are never exposed to bleomycin, this enzyme is present in all mammalian tissues, yeast and some bacteria. The yeast and mammalian forms of BH are highly homologous to each other and to a recently cloned bacterial form. The yeast enzyme is encoded by GAL6 and has amino peptidase, DNA binding and transcriptional regulatory activities. The goal of the proposed work is to discover the cellular functions of this highly conserved bifunctional transcription factor. The first part of the experimental plan proposes a comparative analysis of BH. This effort will entail isolation of the full length mammalian cDNA encoding BH. The DNA binding properties of the mammalian, bacterial (PepC) and yeast enzymes will be compared. Additionally, the spatial localization of the yeast and mammalian enzymes in homologous and heterologous systems will be examined by immunological methods. The second part proposes to uncover physiological roles for BH. This work will take advantage of yeast microbial and molecular genetic methods to identify phenotypes associated with mutation of the GAL6 gene. The applicant suspects that GAL6 may be involved in a mechanism for protecting the cell from the damaging effects of DNA-binding peptides and has designed experiments related to this possibility. Additionally, the investigators will use genetic and biochemical approaches to find genes and proteins that interact functionally and physically with GAL6. Studies are proposed to learn what activities of this DNA-binding- aminopeptidase are important for its transcriptional and other cellular effects. The investigators will take advantage of the X-ray structure to make directed alterations in the protein. The variants will be tested in vivo for their effect on transcriptional activities, bleomycin sensitivity, and any other phenotypes that may be uncovered. Another aim is to clarify the topological features of DNA that are important for GAL6 binding to the GAL1 promoter in vitro and in vivo.
| O'Farrell, P A; Gonzalez, F; Zheng, W et al. (1999) Crystal structure of human bleomycin hydrolase, a self-compartmentalizing cysteine protease. Structure 7:619-27 |
| Zheng, W; Johnston, S A; Joshua-Tor, L (1998) The unusual active site of Gal6/bleomycin hydrolase can act as a carboxypeptidase, aminopeptidase, and peptide ligase. Cell 93:103-9 |
| Zheng, W; Johnston, S A (1998) The nucleic acid binding activity of bleomycin hydrolase is involved in bleomycin detoxification. Mol Cell Biol 18:3580-5 |
| Zheng, W; Xu, H E; Johnston, S A (1997) The cysteine-peptidase bleomycin hydrolase is a member of the galactose regulon in yeast. J Biol Chem 272:30350-5 |
