The Nucleic Acid Binding Activity of Bleomycin Hydrolase Is Involved in Bleomycin Detoxification

Abstract

Yeast bleomycin hydrolase, Gal6p, is a cysteine peptidase that detoxifies the anticancer drug bleomycin. Gal6p is a dual-function protein capable of both nucleic acid binding and peptide cleavage. We now demonstrate that Gal6p exhibits sequence-independent, high-affinity binding to single-stranded DNA, nicked doublestranded DNA, and RNA. A region of the protein that is involved in binding both RNA and DNA substrates is delineated. Immunolocalization reveals that the Gal6 protein is chiefly cytoplasmic and thus may be involved in binding cellular RNAs. Variant Gal6 proteins that fail to bind nucleic acid also exhibit reduced ability to protect cells from bleomycin toxicity, suggesting that the nucleic acid binding activity of Gal6p is important in bleomycin detoxification and may be involved in its normal biological functions.Bleomycin hydrolase is a cysteine peptidase that can cleave the glycopeptide bleomycin, which has been used widely as an anticancer drug in chemotherapy. Bleomycin hydrolase is highly conserved in organisms ranging from bacteria to humans (3, 12). The yeast form of this enzyme has been purified, and its crystal structure has been solved (12). We designated the yeast form of bleomycin hydrolase Gal6, as its expression is regulated by galactose (12, 35). We first identified Gal6p by virtue of its DNA binding property. It binds single-stranded DNA with high affinity (10 nM) and double-stranded DNA with much lower affinity (1 M) (33).The crystal structure of Gal6p revealed that it is a hexamers, its six subunits forming a ring with a central channel of about 20-Å diameter at the entrance. The peptidase active sites are located deep inside this tunnel, sequestered from the exterior. Other nucleic acid-binding proteins with a ring structure, e.g., Escherichia coli DNA polymerase ␤ subunit (16), eukaryotic proliferating cell nuclear antigen (17), and Bacillus subtilis Trp RNA-binding attenuation protein (2), have been identified, as have other proteases with ring structures, e.g., the proteasome and tricorn, which form a unique self-compartmentalizing protease family (18,21). However, Gal6 is the only ring protein with both of these activities.Although a role for Gal6p in bleomycin detoxification has been well documented (7,13,25,33), there is no evidence to date that its nucleic acid binding activity plays a role in this detoxification. The fact that bleomycin itself cleaves nucleic acids (14,28) suggests that the peptidase and nucleic acid binding activities of Gal6p may be functionally linked. On the other hand, the DNA binding activity of Gal6p could be an in vitro artifact and may not reflect a biologically relevant function.We report here the characterization of the nucleic acid binding activity of Gal6p. It binds to single-stranded DNA as well as single-stranded RNA. Immunolocalization demonstrates that most of the Gal6 protein is located in the cytoplasm, indicating that it may function mainly as an RNA-binding protein. Mutation studies show that the ␤-hairpin structure at the opening of th...

Subcellular Localization, Stoichiometry, and Protein Levels of 26 S Proteasome Subunits in Yeast

“…Rabbit anti-yeast cyclophilin antibodies were a gift from Kathryn Sykes. Immunopurified anti-Gal6 antibodies have been described (40). Alkaline phosphatase conjugated goat anti-rabbit and goat antimouse IgG (Bio-Rad) were used for detection in chemifluorescent Western blots.…”

DNA and RNA Binding by the Mitochondrial Lon Protease Is Regulated by Nucleotide and Protein Substrate

“…In yeast, Gal6p has been shown to negatively regulate galactose metabolism and also to hydrolyze bleomycin, which is widely used in cancer chemotherapy. Studies show that DNA-binding mutants of Gal6p fail to protect yeast from bleomycin toxicity (25,26). Recent work has shown that the 19 S component of the 26 S proteasome binds specifically to the GAL1-10 promoter and functions in nucleotide excision repair and transcriptional elongation in yeast (27)(28)(29).…”

MIG1‐dependent and MIG1‐independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p

“…Two genes involved in galactose metabolism, IMP2 and GAL6/BLH1, share the ability to protect cells from the glycopeptide bleomycin (BLM) (Enenkel and Wolf, 1993;Masson and Ramotar, 1996;Zheng and Johnston, 1998). The protease bleomycin hydrolase (Gal6p/Blh1p) is believed to prevent cytotoxic effects by degrading bleomycin.…”

“…In other strains significant differences between the wild-type strain and the gal6 mutant were observed in survival to short-term treatments with bleomycin (Zheng & Johnston, 1998). We then performed the analysis under similar experimental conditions ( Figure 4C).…”