Nuclear deoxyribonucleic acid polymerase. Purification and properties of the homogeneous enzyme from human KB cells.
Nuclear deoxyribonucleic acid polymerase. Purification and properties of the homogeneous enzyme from human KB cells.
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核脱氧核糖核酸聚合酶。
DOI：
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发表时间：
1974
影响因子：
4.8
通讯作者：
D. Korn
中科院分区：
文献类型：
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作者：
T. S. Wang;W. D. Sedwick;D. Korn
我们从培养的KB细胞中纯化了小核DNA聚合酶，纯度≥95%。在十二烷基硫酸钠聚丙烯酰胺凝胶板上的电泳表明，聚合酶由分子量为43，000的单链多肽链组成，并且该大小与通过凝胶过滤测定的聚合酶活性的大小一致。尽管该酶进行常规聚合反应，在3 '-羟基末端掺入互补的脱氧核苷三磷酸，伴随着PPi的化学计量释放，但不能证明其进行PPi交换。聚合酶缺乏核酸外切酶活性，特别是3 '→ 5'-核酸外切酶活性，如通过最灵敏的可用测定所测试的。然而，该酶确实表现出显著的引物模板依赖性的脱氧核苷三磷酸到脱氧核苷一磷酸的转化。我们提出，这种反应代表了一种独立的聚合酶相关活性，与3 '→ 5'-外切核酸酶无关，与从原核聚合酶研究中开发的模型相反。最后，KB聚合酶不能从合成的均聚物引物-模板切除错配的引物末端，而是可以利用这样的末端作为聚合的功能性起始位点。
Abstract We have purified the small nuclear DNA polymerase from cultured KB cells to ≥95% homogeneity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gel slabs demonstrates that the polymerase is comprised of a single polypeptide chain of molecular weight 43,000, and this size is in agreement with that of the polymerase activity as determined by gel filtration. Although the enzyme carries out a conventional polymerization reaction, incorporating complementary deoxynucleoside triphosphates at 3'-hydroxyl termini with concomitant stoichiometric release of PPi, it cannot be demonstrated to carry out PPi exchange. The polymerase is devoid of exonuclease activities, particularly the 3'→5'-exonuclease activity, as tested by the most sensitive available assays. However, the enzyme does exhibit prominent primer-template-dependent conversion of deoxynucleoside triphosphate to deoxynucleoside monophosphate. We propose that this reaction represents an independent polymerase-associated activity that is unrelated to 3'→5'-exonuclease, in contrast to the model that has been developed from studies with prokaryotic polymerases. Finally, the KB polymerase is unable to excise mismatched primer termini from a synthetic homopolymer primer-template, but rather can utilize such termini as functional initiation sites for polymerization.