Atrazhev A, Zhang S, Grosse F
German Primate Center, Department of Virology and Immunology, Göttingen.
Eur J Biochem. 1992 Dec 15;210(3):855-65. doi: 10.1111/j.1432-1033.1992.tb17489.x.
A binding protein for single-stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE-cellulose, blue-Sepharose, ssDNA-cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s = 5.1 S) and gel filtration (Stokes' radius approximately 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1:1:1 stoichiometry for the 70 + 55/30/11-kDa complex. The ssDNA binding protein (SSB) covered approximately 20-25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA-polymerase-alpha-primase complex was stimulated by the ssDNA binding protein 1.2-fold on poly(dA).(dT)14 and 10-13-fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase-primase was not affected on poly(dA).(dT)14; with poly(dT).(rA)10 an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase alpha to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300-600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20-120 nucleotides when SSB was absent. DNA-primase-initiated DNA replication on M13 DNA was inhibited by SSB in a concentration-dependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.
通过在DEAE - 纤维素、蓝色琼脂糖凝胶、单链DNA - 纤维素和快速蛋白质液相色谱法（FPLC）Mono Q柱上进行层析，从牛胸腺中纯化出一种单链DNA（ssDNA）结合蛋白，使其纯度接近均一。最纯的组分由四种分子量分别为70、55、30和11 kDa的多肽组成。分子量为55 kDa的多肽很可能是最大多肽的降解形式。该复合物在甘油梯度超速离心（沉降系数s = 5.1 S）和凝胶过滤（斯托克斯半径约为5.1 nm）中均作为一个整体迁移。综合这些数据表明其天然分子量约为110 kDa，这与70 + 55/30/11 kDa复合物的1:1:1化学计量比相符。从凝胶迁移实验和DNase I消化研究均表明，ssDNA结合蛋白（SSB）在M13mp8 ssDNA上覆盖约20 - 25个核苷酸。同源的DNA - 聚合酶α - 引发酶复合物在poly(dA)·(dT)14上被ssDNA结合蛋白刺激1.2倍，在单引物的M13mp8 DNA上被刺激10 - 13倍。刺激主要是由于通过稳定的二级结构促进了DNA合成，这表现为许多（但不是全部）暂停位点的消失。聚合酶 - 引发酶在poly(dA)·(dT)14上的持续合成能力未受影响；在poly(dT)·(rA)10存在SSB时，观察到产物长度增加约两倍。这种增加归因于聚合酶α更容易重新结合到已完成的DNA片段上，而不是聚合酶内在持续合成能力的增强。同样，在单引物的M13 DNA上，存在SSB时形成了300 - 600个核苷酸长的产物，而不存在SSB时则为20 - 120个核苷酸。SSB以浓度依赖的方式抑制M13 DNA上由DNA - 引发酶起始的DNA复制。然而，由于起始RNA引物的位点较少，形成了更均一的产物。
