Wang Z F, Yang J, Nie Z Q, Wu M
Department of Biological Sciences, University of Maryland Baltimore County 21228.
Biochemistry. 1991 Jan 29;30(4):1127-31. doi: 10.1021/bi00218a034.
A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.
一种粗体外系统可在电子显微镜定位的D-环位点附近启动叶绿体DNA合成[吴，M.，娄，J.K.，张，D.Y.，张，C.H.，&聂，Z.Q.（1986年）美国国家科学院院刊83，6761 - 6765]，该系统由可溶性蛋白质和从纯化类囊体膜中提取的蛋白质组成。在本文中，通过在肝素 - 琼脂糖、DEAE - 纤维素和单链DNA - 琼脂糖柱上的连续色谱分离以及在甘油梯度中的沉降，从该体外系统的可溶性蛋白质部分纯化出一种DNA聚合酶活性，使其接近均一。在甘油梯度中，酶活性沉降在对应于110 kDa蛋白质的位置。在SDS - 聚丙烯酰胺凝胶上对高度纯化的部分进行电泳分析，显示出一条主要的多肽带，其表观分子量约为116 kDa。原位DNA聚合酶活性测定表明，DNA聚合功能与116 kDa带和一个80 kDa带相关，80 kDa带可能是该酶的一个亚基。聚合活性受到N - 乙基马来酰亚胺、溴化乙锭和双脱氧胞嘧啶三磷酸的抑制，并且对阿非科林相对耐药。聚（dA）·（dT）10和带缺口的双链DNA是优选的模板。纯化的酶不具有外切核酸酶活性，并且可以在含有叶绿体DNA复制起点 的超螺旋质粒DNA模板中启动DNA复制。
