Involvement of Chromosome in Regulation of DNA Replication and Repair

我们已经开发了一个用于SV40微小染色体复制的无Call系统，由于这个重组系统缺乏从头开始的核小体组装活性，所以我们可以特异性地检测双亲组蛋白在染色体复制过程中的行为。溶液杂交分析表明，组蛋白并未完全从模板DNA中分离出来，但在某种程度上与模板DNA有关。用链特异性探针和MNase消化新生的单核细胞DNA得到的结果表明，在用纯化的蛋白质进行染色体复制的过程中，双亲的组蛋白八聚体分布在前导和滞后链之间。当用TopoⅡ单独作为旋转酶复制SV40染色体时，添加Topo II的反应产生了较短的前导链，但成熟大小的链在添加Topo II的反应中积累。这些结果表明，Topo II在…的SV40染色体复制的后期起着关键的旋转酶作用另一方面，我们开发了一种无细胞系统，支持组织成核小体的DNA，即SV40染色体的修复反应。标准反应混合物包括紫外线照射的SV40染色体、未照射的质粒DNA、HeLa细胞的全细胞提取物(曼利提取物)、ATP再生系统和包括[α-^&lt；32&gt；P]dCTP在内的几种低分子物质。30ﾟC孵育后，提取DNA，EcoRI消化，琼脂糖凝胶电泳法分离。放射自显影检测DNA的修复合成和缺口翻译。SV40DNA的DNA合成依赖于照射后DNA的剂量。当我们用XP-A或XP-C细胞提取液替代HeLa细胞提取液时，依赖紫外线的DNA合成显著降低。XP-A和XP-C细胞提取物的混合使DNA合成恢复到与HeLa细胞提取物相似的水平。因此，我们的体外系统似乎对重建染色体DNA切除修复的至少一些特征是有用的。较少

We have developed a call-free system for replication of SV40 minichromosomes with purified proteins.Since this reconstituted system lacked activity of de novo nucleosome assembly, we could specifically examine the behavior of parental histones during chromosome replication. Solution hybridization analyses suggested that the histones were not completely detached from but somehow associated with template DNA. Using strand-specific probes and MNase digested nascent mononucleosomal DNA, we obtained the results indicating that the parental histone octamers were segregated distributively between leading and lagging strands during the chromosome replication with purified proteifis.When SV40 chromosomes were replicated with topoI alone as a swivelase, the reaction produced shorter leading strands but those of mature size were accumulated in the reaction supplemented with topo II. These results indicate that topo II has a crucial role as a swivelase in the late stage of SV40 chromosome replicat … More ion in vitro.On the other hand, we have developed a cell-free system supporting repair reactions on DNA organized into hucleosomes, that is, SV40 chromosomes. Standard reaction mixtures contained UV-irradiated SV40 chromosomes, unirradiated plasmid DNA, whole cell extracts of HeLa cells(Manley's extracts), ATP-regenerating system and several low molecular weight materials including[alpha-^<32>P]dCTP. After incubated at 30ﾟC, DNAs were isolated, digested with EcoRI and separated by agarose gel electrophoresis. Repair synthesis as well as nick-translation of DNAs were detected by autoradiography. DNA synthesis on SV40 DNA was dependent on doses of irradiated DNA. When we used cell extracts made from XP-A or XP-C cells in place of HeLa cell extracts, the UV-dependent DNA synthesis decreased remarkably. Mixing of XP-A and XP-C cell extracts restored the DNA synthesis to similar level as HeLa cell extracts. Thus our in vitro system appears to be useful for reconstituting at least some features of chromosomal DNA excision repair. Less