Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.
Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.
复制标题
体外核 DNA 合成是通过体内以时间特定方式组装的稳定复制叉介导的。
DOI：
10.1128/mcb.8.5.1923-1931.1988
复制
发表时间：
1988
影响因子：
5.3
通讯作者：
Stillman,BW
中科院分区：
文献类型：
--
作者：
Heintz,NH;Stillman,BW
一种无细胞核复制系统，它是S期特异性的，需要DNA聚合酶α的活性，并且被来自S期细胞的胞质因子刺激3 - 8倍，用于检查体外染色体DNA合成的时间特异性。通过检查来自甲氨蝶呤耐药CHOC 400细胞的扩增的200-脱氢酶二氢叶酸还原酶结构域的限制性片段的复制作为细胞周期的函数，直接评估分离的细胞核中DNA合成的时间特异性。在细胞核制备收集在G1/S边界的细胞周期中，扩增序列的合成开始在直接二氢叶酸还原酶的起源区域和延长持续60至80分钟。扩增的限制性片段在细胞核中从早期S期细胞在体外的合成顺序似乎是无法区分的，从体内。从稍后在S期稳定的CHOC 400细胞制备的细胞核合成了其他扩增片段的特征子集。片段标记模式的特异性在4°C下短期储存是稳定的。刺激因子在胞质提取物中的发生是细胞周期依赖性的，在早期G1期提取物中观察到最小的刺激，而在S期细胞的胞质提取物中观察到最大的刺激。在G1期细胞的细胞核中没有观察到染色体合成，S期细胞的胞质提取物也没有诱导G1期细胞核中的染色体复制。与染色体DNA合成相反，线粒体DNA在体外的复制不受细胞质因子的刺激，并且在整个G1期和S期以相等的速率发生。这些研究表明，在分离的细胞核中的染色体DNA复制是由稳定的复制叉介导的，这些复制叉在体内以时间特异性的方式组装，并表明在体外观察到的合成机制准确地反映了体内的合成机制。
A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase α, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4°C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.