Skalski V, Liu S H, Cheng Y C
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA.
Biochem Pharmacol. 1995 Sep 7;50(6):815-21. doi: 10.1016/0006-2952(95)00205-e.
A 3'-->5' exonuclease has been highly purified from the cytosol of human acute lymphoblastic leukemia H9 cells. The apparent molecular weight of this enzyme was approximately 50,000, as indicated by its sedimentation in glycerol gradients. The exonuclease did not copurify with DNA polymerase activity, required MgCl2 for its exonucleolytic activity, and was inhibited by KCl above 60 mM. The enzyme was active on single-stranded DNA, DNA duplexes and DNA/RNA duplexes, and it was efficient at removing 3'-terminal mispairs from DNA. The products of the exonucleolytic reaction were deoxynucleoside 5'-monophosphates. The behavior of the exonuclease was examined on DNA terminated at the 3' end with a variety of dideoxynucleosides that are potent against human immunodeficiency virus type 1. The exonuclease has a broad substrate specificity; however, the rate of the enzymatic reaction varied among the D dideoxynucleosides tested (ddAMP = ddCMP > d4TMP > AZTMP). Similarly, the enzyme was examined for its reactivity with DNA terminated by either the D or L enantiomers of ddC, SddC or FddC. The removal of analogs with the native D configuration was at least 6-fold more rapid than that of the L-compounds, and the type of structural modification had an impact on the rate at which the D enantiomers were removed (SddCMP > ddCMP > FddCMP). The monophosphate forms of AZT, D4T, L-FddC and L-ddC were potent inhibitors of the exonuclease at micromolar concentrations, while D-ddCMP partially inhibited the enzyme at millimolar concentrations. Based on its physical and enzymatic properties, this exonuclease represents a novel enzyme that may have an important role in determining the relative potencies of dideoxynucleosides against human immunodeficiency virus type 1.
一种3'→5'核酸外切酶已从人急性淋巴细胞白血病H9细胞的胞质溶胶中高度纯化出来。通过其在甘油梯度中的沉降显示，这种酶的表观分子量约为50,000。该核酸外切酶与DNA聚合酶活性不共纯化，其核酸外切活性需要MgCl2，并且在60 mM以上的KCl存在时受到抑制。该酶对单链DNA、DNA双链体和DNA/RNA双链体均有活性，并且能有效地从DNA中去除3'末端错配。核酸外切反应的产物是脱氧核苷5'-单磷酸。用多种对人免疫缺陷病毒1型有效的双脱氧核苷在3'末端终止的DNA上检测了该核酸外切酶的行为。该核酸外切酶具有广泛的底物特异性；然而，在所测试的双脱氧核苷（ddAMP = ddCMP > d4TMP > AZTMP）中，酶促反应的速率有所不同。同样，检测了该酶与由ddC的D或L对映体、SddC或FddC终止的DNA的反应性。去除具有天然D构型的类似物的速度至少比L-化合物快6倍，并且结构修饰的类型对D对映体的去除速率有影响（SddCMP > ddCMP > FddCMP）。AZT、D4T、L-FddC和L-ddC的单磷酸形式在微摩尔浓度下是该核酸外切酶的有效抑制剂，而D-ddCMP在毫摩尔浓度下部分抑制该酶。基于其物理和酶学性质，这种核酸外切酶代表了一种新型酶，可能在确定双脱氧核苷对人免疫缺陷病毒1型的相对效力方面起重要作用。
