A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata
A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata
复制标题
DOI：
10.1093/nar/26.20.4733
复制
发表时间：
1998-10-15
影响因子：
14.9
通讯作者：
Ray, DS
中科院分区：
文献类型：
--
作者：
Engel, ML;Ray, DS
束状锥虫的线粒体DNA（动基体DNA）由微环和大环拓扑地互锁在每个细胞的单个网络中组成。单个小环从位于小环DNA上相隔180度的两个复制起点中的任一个单向复制。小环前导链合成的起始涉及RNA引物的合成，该RNA引物在复制的最后阶段被去除。我们在这里报告的纯化，以接近同质的结构特异性DNA内切酶的基础上的RNase H活性的酶的聚（rA）。聚（dT）衬底。全细胞和动基体提取物的RNase H活性凝胶分析表明，该酶富集在动基体组分中。该酶的DNA核酸内切酶活性对退火至模板链的DNA引物具有特异性，并且需要未退火的5'尾。该酶切割第一个碱基配对核苷酸的3'，释放完整的尾部。纯化的酶在SDS凝胶上以32 kDa蛋白迁移，斯托克半径为21.5埃，沉降系数为3.7 s，表明该蛋白在溶液中是天然分子量为32.4 kDa的单体。这些结果表明，酶可能参与在小环复制过程中的RNA引物去除。
The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endonuclease based on the RNase H activity of the enzyme on a poly(rA). poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 Angstrom and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.