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Title
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Yeast bleomycin hydrolase is a DNA-binding cysteine protease. Identification, purification, biochemical characterization.
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Authors
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H.E.Xu,
S.A.Johnston.
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Ref.
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J Biol Chem, 1994,
269,
21177-21183.
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PubMed id
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Abstract
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Bleomycin (BLM) is a DNA binding and damaging antibiotic produced by
Streptomyces verticillus that has been used as an anti-tumor drug for various
human cancers. The mammalian BLM hydrolase is a cysteine protease that
inactivates BLM to relieve the toxicity of BLM to normal and tumor cells. The
normal physiological function of BLM hydrolase is not known, but its activity
limits the use of BLM in cancer chemotherapy. We have discovered a DNA binding
activity for the yeast homolog of the mammalian BLM hydrolase in the course of
studying the interaction of GAL4, a DNA-binding transcription factor, with its
DNA recognition sites. Using gel mobility shift assays, we have purified a
protein from yeast that binds specifically to the GAL4 DNA-binding sites. The
purified protein is a tetramer of a 48-kDa polypeptide. The gene encoding the
48-kDa polypeptide was cloned and has a high homology to rabbit BLM hydrolase.
The purified protein was confirmed to have a cysteine protease activity that can
hydrolyze and inactivate BLM in vitro. We have established the optimal
conditions for the protease activity of this protein and biochemically
characterized its DNA binding activity. This protein binds both single- and
double-stranded forms of the GAL4 DNA-binding sites with high affinity (10 nM to
single strand and 1 microM to double strand). Its DNA binding activity is heat
stable and resistant to various detergents. This protein may represent the first
example of a eukaryotic DNA-binding protease. The discovery of a DNA binding
activity for BLM hydrolase suggests an in vivo interaction between it and BLM on
DNA.
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