Li C, Englund P T
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 1997 Aug 15;272(33):20787-92. doi: 10.1074/jbc.272.33.20787.
We have purified to near homogeneity a DNA primase from a mitochondrial fraction of the trypanosomatid Crithidia fasciculata. The enzyme is a single polypeptide chain of 28 kDa. Using a poly(dT) template and ATP as a substrate, the enzyme makes oligonucleotides of which the vast majority are about 10 nucleotides in size or smaller. With a single-stranded M13 DNA template and the four rNTPs as substrates, the enzyme makes heterogeneous oligonucleotides in the same size range. These oligonucleotides efficiently prime the synthesis of DNA by the Klenow DNA polymerase. Immunolocalization with antibodies against the purified enzyme confirms that the primase is mitochondrial. Furthermore, the enzyme localizes to specific regions of the cell's single mitochondrion, above and below the condensed kinetoplast DNA. The primase does not co-localize with the mitochondrial topoisomerase II and DNA polymerase beta, both of which are associated with two protein complexes positioned on opposite sides of the kinetoplast disc. These localization studies have significant implications for the mechanism of kinetoplast DNA replication.
我们已从锥虫克氏锥虫的线粒体组分中纯化出一种DNA引发酶，纯度近乎均一。该酶是一条28 kDa的单多肽链。以聚（dT）模板和ATP作为底物时，该酶合成的寡核苷酸绝大多数长度约为10个核苷酸或更短。以单链M13 DNA模板和四种rNTP作为底物时，该酶合成相同大小范围内的异质寡核苷酸。这些寡核苷酸能有效引发Klenow DNA聚合酶对DNA的合成。用针对纯化酶的抗体进行免疫定位证实该引发酶定位于线粒体。此外，该酶定位于细胞单个线粒体的特定区域，位于浓缩的动基体DNA的上方和下方。引发酶与线粒体拓扑异构酶II和DNA聚合酶β不共定位，这两种酶都与位于动基体盘两侧的两个蛋白质复合物相关。这些定位研究对动基体DNA复制机制具有重要意义。
