摘要
Activities of trimalonic acid fullerene (TMA C_60) on DNA restrictive enzymatic reaction were investigated by using two restrictive endonucleases Hind III and EcoR I and plasmid pEGFP-N1 with single restric-tive site for both enzymes. Meanwhile, TMA C60 was also tested to clarify its effects on polymerase chain reaction (PCR) with the catalyst of Taq DNA polymerase and the template of plasmid pEGFP-N1. The products from restrictive reactions or PCR were detected by agarose gel electrophoresis. It was found that the product amounts from restrictive reactions or PCR decreased significantly with addition of TMA C60. The inhibition by TMA C60 was dose-dependent and IC50 values for reactions of Hind III, EcoR I and PCR were 16.3, 6.0 and 6.0 μmol/L, respectively. Addition of two scavengers of reactive oxygen species (ROS), L-ascorbic acid-2-phosphate ester magnesium and sodium azide at the con-centrations of 2―10 mmol/L did not antagonize the activities of TMA C60 against PCR and two restrictive reactions. However, increase of Taq DNA polymerase amounts in PCR system antagonized the activities of TMA C60. These data implied that TMA C60 was able to inhibit the activities of the three above-mentioned enzymes involved in DNA metabolism, and that this inhibition probably did not correlate to ROS.
Activities of trimalonic acid fullerene (TMA C60) on DNA restrictive enzymatic reaction were investigated by using two restrictive endonucleases Hind III and EcoR I and plasmid pEGFP-N1 with single restrictive site for both enzymes. Meanwhile, TMA C60 was also tested to clarify its effects on polymerase chain reaction (PCR) with the catalyst of Taq DNA polymerase and the template of plasmid pEGFP-N1. The products from restrictive reactions or PCR were detected by agarose gel electrophoresis. It was found that the product amounts from restrictive reactions or PCR decreased significantly with addition of TMA C60. The inhibition by TMA C60 was dose-dependent and IC50 values for reactions of Hind III, EcoRI and PCR were 16.3, 6.0 and 6.0 tJmol/L, respectively. Addition of two scavengers of reactive oxygen species (ROS), L-ascorbic acid-2-phosphate ester magnesium and sodium azide at the concentrations of 2-10 mmol/L did not antagonize the activities of TMA C60 against PCR and two restrictive reactions. However, increase of Taq DNA polymerase amounts in PCR system antagonized the activities of TMA C60. These data implied that TMA C60 was able to inhibit the activities of the three above-mentioned enzymes involved in DNA metabolism, and that this inhibition probably did not correlate to ROS.
基金
the Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20030007011)
the Basic Research Foundation of Beijing Institute of Technology (Grant No. 000Y06)
关键词
内切核苷酸
抑制作用
DNA
聚合酶
氧化
trimalonic acid C60 (TMA C60), DNA restrictive endonucleases, Taq DNA polymerase, reactive oxygen species, inhibition