MECHANISM OF MITOCHONDRIAL-DNA REPLICATION IN MOUSE L-CELLS - KINETICS OF SYNTHESIS AND TURNOVER OF INITIATION SEQUENCE
MECHANISM OF MITOCHONDRIAL-DNA REPLICATION IN MOUSE L-CELLS - KINETICS OF SYNTHESIS AND TURNOVER OF INITIATION SEQUENCE
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DOI：
10.1016/0022-2836(78)90269-3
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发表时间：
1978-01-01
影响因子：
5.6
通讯作者：
CLAYTON, DA
中科院分区：
文献类型：
--
作者：
BOGENHAGEN, D;CLAYTON, DA
使用胸苷激酶加小鼠LA 9 [肿瘤成纤维细胞]细胞的脉冲追踪放射性标记实验表明，线粒体DNA的7 S线粒体DNA起始序列的合成和翻转速度比先前确定的更快。这些脉冲追踪标记实验还确定了小鼠LA 9细胞线粒体DNA的复制时间为1 h。脉冲标记的7 S线粒体DNA起始序列的半衰期约为70分钟。这种周转是如此之快，至少95%的线粒体DNA起始序列合成的损失营业额没有作为引物扩增合成的线粒体DNA重链。7S线粒体DNA更新的机制并不导致线粒体内游离7S线粒体DNA单链的显著积累。7S线粒体DNA起始序列的再合成足够快，使得大多数线粒体DNA分子保持为置换环分子。聚合成线粒体DNA的所有核苷酸中约20%掺入7S起始序列。新合成的7S线粒体DNA链的大小从大约500-620个核苷酸变化。聚丙烯酰胺/尿素凝胶电泳可分辨几个大小类别，每个类别具有大致相同的周转率。小鼠LD细胞保持其线粒体DNA基因组是单环的，头到尾的二聚体。由于这些单环二聚体的显著部分仅含有1个置换环，如果链的合成受到超螺旋形成的自由能的限制，则此类分子中起始序列的大小应该是其两倍长。与小鼠LA 9细胞相比，从该细胞系获得了相同的7条S链大小类别的阵列。这表明7S线粒体DNA合成的程度很可能是由核苷酸序列特异性事件决定的。
Pulse-chase radioactive labeling experiments using thymidine kinase-plus mouse LA9 [neoplastic fibroblast] cells have shown that the 7 S mitochondrial DNA initiation sequence of mitochondrial DNA is synthesized and turned over at a faster rate than previously determined. These pulse-chase labeling experiments have also determined that the replication time of mouse LA9 cell mitochondrial DNA is 1 h. The half-life of pulse-labeled 7 S mitochondrial DNA initiation sequences is approximately 70 min. This turnover is so rapid that at least 95% of the mitochondrial DNA initiation sequences synthesized are lost to turnover without acting as primers for expansion synthesis of the mitochondrial DNA heavy strand. The mechanism of 7 S mitochondrial DNA turnover does not lead to significant accumulation of free 7 S mitochondrial DNA single-strands within mitochondria. Resynthesis of the 7 S mitochondrial DNA initiation sequence is sufficiently rapid that the majority of mitochondrial DNA molecules are maintained as displacement loop molecules. Approximately 20% of all nucleotides polymerized into mitochondrial DNA are incorporated into the 7 S initiation sequences. The size of newly synthesized 7 S mitochondrial DNA strands varies from about 500-620 nucleotides. Several size classes are resolved by polyacrylamide/urea gel electrophoresis and each class has approximately the same turnover rate. Mouse LD cells maintain their mitochondrial DNA genomes are unicircular, head-to-tail dimers. Since a significant fraction of these unicircular dimers contain only 1 displacement loop, the size of the initiation sequence in such molecules should be twice as long if synthesis of the strand is limited by the free energy of superhelix formation. An identical array of size classes of 7 S strands is obtained from this cell line as compared to mouse LA9 cells. This indicates that the extent of 7 S mitochondrial DNA synthesis is most likely determined by a nucleotide sequence specific event.