Rose Bengal mediated inhibition of DNA polymerases: mechanism of inhibition of avian myeloblastosis virus reverse transcriptase under photooxidative conditions.
Rose Bengal mediated inhibition of DNA polymerases: mechanism of inhibition of avian myeloblastosis virus reverse transcriptase under photooxidative conditions.
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DOI：
10.1021/bi00278a035
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发表时间：
1982-09
期刊：
影响因子：
2.9
通讯作者：
S. Srivastava;M. Modak
中科院分区：
文献类型：
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作者：
S. Srivastava;M. Modak
原核、真核和肿瘤病毒来源的DNA聚合酶在光照条件下暴露于虎红后不可逆地失活（光氧化条件）。这些酶在黑暗（非氧化）条件下的失活是完全可逆的[Srivastava，S. K.，& Modak，M. J.（1982）Biochemistry 21，4633-4639]。发现光氧化对酶-虎红复合物的主要影响是暴露于光5分钟内模板-引物结合能力的丧失。模板引物而非底物脱氧核苷酸的存在一致地提供了对虎红介导的光氧化灭活的部分保护。在这些条件下，虎红未解离预先形成的酶-模板-引物复合物。然而，在存在虎红的情况下，该复合物长时间暴露于光（25-30 min）导致催化活性完全失活，而不影响酶与模板引物结合的能力。这些结果清楚地表明，在虎红存在下，禽成髓细胞瘤病毒逆转录酶的氧化灭活涉及酶内的一个结构域，该结构域含有模板-引物结合位点以及逆转录酶聚合和核酸酶活性表达所需的额外位点。
DNA polymerases from prokaryotic, eukaryotic, and oncornaviral sources are irreversibly inactivated upon exposure to Rose Bengal in the presence of light (photooxidation conditions). Inactivation of these enzymes under dark (nonoxidative) conditions is totally reversible [Srivastava, S. K., & Modak, M. J. (1982) Biochemistry 21, 4633-4639]. The primary effect of photooxidation on the enzyme-Rose Bengal complex was found to be the loss of template-primer binding ability within 5 min of exposure to light. The presence of template-primer but not the substrate deoxynucleotides consistently provided partial protection against Rose Bengal mediated photooxidative inactivation. Preformed enzyme-template-primer complexes were not dissociated by Rose Bengal under these conditions. However, prolonged exposure (25-30 min) of this complex to light in the presence of Rose Bengal led to complete inactivation of catalytic activity without affecting the ability of enzyme to bind to template-primer. These results clearly indicate that oxidative inactivation of avian myeloblastosis virus reverse transcriptase in the presence of Rose Bengal involves a domain within the enzyme that contains the template-primer binding site as well as an additional site which is required for the expression of both the polymerization and nuclease activities of the reverse transcriptase.