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|Título:||Alteration of the conserved residue tyrosine-158 to histidine renders human O6-alkylguanine-DNA alkyltransferase insensitive to the inhibitor O6- benzylguanine|
|Autor:||Xu Welliver, M.|
Leitão, J. M.
Pegg, A. E.
|Resumo:||The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from alkylation damage. O6-Benzylguanine (BG) is a potent inactivator of human AGT (ED50 of 0.1 μM) that is currently undergoing clinical trials to enhance chemotherapy by alkylating agents. In a screen of AGT mutants randomly mutated at position glycine-160, we found that the double mutant Y158H/G160A protected Escherichia coli from killing by N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) even in the presence of BG and that the AGT activity of this mutant was strongly resistant to BG (ED50 of 180 μM). Because the single mutant G160A was not resistant to BG, this suggested that the presence of the charged histidine residue at position 158 was responsible. This hypothesis was confirmed by the construction of the single mutation Y158H. The Y158H-mutant AGT was slightly less active than wild-type AGT for the repair of mcthylated DNA in vitro, but it protected E. coli from killing by MNNG even in the presence of BG and had an ED50 for the inactivation by BG of 620 μM. In contrast, mutant Y158F had an ED50 of 0.2 μM. Previous studies (M. Xu-Welliver et al., Cancer Res., 58: 1936-1945, 1998) have shown that mutant P140K is highly resistant to BG (ED50 of > 1200 μM). Models of human AGT suggest that the side chain of the lysine inserted into this mutant is close to tyrosine-158 and that the positively charged lysine side-chain may interfere with BG binding. The double mutants P140K/Y158H and P140K/Y158F resembled P140K and Y158H in being highly resistant to BG, but the use of a sensitive assay for reaction of BG with AGT indicated that their abilities to react were in the order P140K/Y158H < P140K < P140K/Y158F. These results confirm that the presence of a positively charged residue close to the active site of human AGT renders it highly resistant to BG without substantially affecting activity toward methylated DNA substrates. Such mutants may limit the value of BG therapy if they arise in malignant cells during chemotherapy, but the mutant sequences may be useful for gene therapy approaches in which BG-resistant human AGTs are used to prevent hematopoietic toxicity. At least 28 AGT sequences (from 25 species) have now been described. In 25 of these, the position equivalent to 158 in the human AGT is also a tyrosine, and in the other 3, it is a phenylalanine. The importance of an aromatic ring side chain at this position is emphasized by previous studies (S. Edara et al., Carcinogenesis, 16: 1637- 1642, 1995), which show that the replacement by alanine renders human AGT inactive. Our results show that histidine can also substitute for tyrosine at this position.|
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|Cancer Res-1999-Xu-Welliver-1514-9.pdf||726,23 kB||Adobe PDF||Ver/Abrir|
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