Lin H J, Cannon G C, Heinhorst S
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg 39406.
Nucleic Acids Res. 1990 Nov 25;18(22):6659-63. doi: 10.1093/nar/18.22.6659.
A DNA polymerase has been highly purified from Anacystis nidulans R2. Electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gels revealed that the final fraction contains three bands of Mr 107,000, 93,000, and 51,000, respectively. Analysis of purified DNA polymerase activity in situ indicates that of the three polypeptides the Mr 107,000 species has the catalytic activities. The native molecular weight of the enzyme was estimated by glycerol gradient sedimentation to be 100,000. The enzyme has an absolute requirement for a divalent cation. Mg2+ can be replaced with Mn2+, but the DNA polymerase is less active. Potassium chloride stimulates the enzyme, while potassium phosphate has no apparent effect. The enzyme is active over a pH range from 7.5 to 9.5 in 50mM Tris-HCl buffer. The ability of the cyanobacterial DNA polymerase to use activated DNA as a template, its associated 3'----5' and 5'----3' exonuclease activities, as well as its resistance to N-ethylmaleimide, dideoxynucleotides, arabinosyl-CTP and aphidicolin suggest a similarity between this enzyme and E. coli DNA polymerase I. This is the first characterization of a DNA polymerase from a cyanobacterium.
已从集胞藻6803（Anacystis nidulans R2）中高度纯化出一种DNA聚合酶。在十二烷基硫酸钠-聚丙烯酰胺凝胶中的电泳分析表明，最终级分分别含有分子量为107,000、93,000和51,000的三条带。对原位纯化的DNA聚合酶活性的分析表明，在这三种多肽中，分子量为107,000的物种具有催化活性。通过甘油梯度沉降估计该酶的天然分子量为100,000。该酶对二价阳离子有绝对需求。Mg2+可以被Mn2+替代，但DNA聚合酶的活性较低。氯化钾刺激该酶，而磷酸钾没有明显作用。在50mM Tris-HCl缓冲液中，该酶在pH 7.5至9.5的范围内具有活性。蓝藻DNA聚合酶使用活化DNA作为模板的能力、其相关的3'→5'和5'→3'核酸外切酶活性，以及其对N-乙基马来酰亚胺、双脱氧核苷酸、阿拉伯糖基-CTP和阿非迪霉素的抗性表明该酶与大肠杆菌DNA聚合酶I之间存在相似性。这是首次对来自蓝藻的DNA聚合酶进行表征。
