Seki M, Enomoto T, Eki T, Miyajima A, Murakami Y, Hanaoka F, Ui M
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Biochemistry. 1990 Jan 30;29(4):1003-9. doi: 10.1021/bi00456a024.
Polyoma virus large tumor antigen (PyV T antigen) has been purified to near homogeneity by immunoaffinity column chromatography. We have detected DNA helicase and ATPase (nucleoside-5'-triphosphatase) activities in the purified PyV T antigen fraction and characterized these activities. The ATPase activity was stimulated about 2-fold by poly(dT), which was the most effective stimulator among the synthetic polynucleotides tested. Natural nucleic acids, such as calf thymus native and heat-denatured DNA, and single-stranded circular fd DNA were also effective, but the degree of stimulation was less than 1.5-fold. The basal and poly(dT)-stimulated ATPase activities showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optima. The preference for nucleoside 5'-triphosphates was ATP, dATP greater than CTP, UTP much greater than GTP. The only difference observed between the two activities was salt sensitivity. The basal ATPase activity was resistant to KC1 up to 300 mM. In contrast, poly-(dT)-stimulated activity was reduced to the level of basal activity at 300 mM KC1. DNA helicase activity required divalent cations and was dependent on hydrolysis of ATP. The activity showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optimum as the two ATPase activities, and the salt sensitivity of DNA helicase activity was similar to that of poly(dT)-stimulated ATPase activity. The helicase activity was inhibited competitively by the addition of single-stranded or double-stranded DNA, and a relatively high inhibitory activity was observed with poly [d(A-T)]. The PyV T antigen helicase was found to migrate in the 3' to 5' direction along the DNA strand to which the protein bound.
多瘤病毒大肿瘤抗原（PyV T抗原）已通过免疫亲和柱层析纯化至近乎同质。我们在纯化的PyV T抗原组分中检测到了DNA解旋酶和ATP酶（核苷-5'-三磷酸酶）活性，并对这些活性进行了表征。ATP酶活性被聚（dT）刺激了约2倍，聚（dT）是所测试的合成多核苷酸中最有效的刺激剂。天然核酸，如小牛胸腺天然和热变性DNA以及单链环状fd DNA也有效，但刺激程度小于1.5倍。基础和聚（dT）刺激的ATP酶活性对核苷5'-三磷酸具有相似的偏好、对二价阳离子的需求以及最适pH值。对核苷5'-三磷酸的偏好是ATP＞dATP＞CTP，UTP远大于GTP。观察到的这两种活性之间的唯一差异是盐敏感性。基础ATP酶活性对高达300 mM的KCl具有抗性。相比之下，聚（dT）刺激的活性在300 mM KCl时降低至基础活性水平。DNA解旋酶活性需要二价阳离子，并且依赖于ATP的水解。该活性对核苷5'-三磷酸的偏好、对二价阳离子的需求以及最适pH值与两种ATP酶活性相似，并且DNA解旋酶活性的盐敏感性与聚（dT）刺激的ATP酶活性相似。通过添加单链或双链DNA竞争性抑制解旋酶活性，并且观察到聚[d（A-T）]具有相对较高的抑制活性。发现PyV T抗原解旋酶沿着与蛋白质结合的DNA链从3'向5'方向迁移。
