Roles of regulation of DNA replication in genomic imprinting

染色质的表观遗传修饰在基因组印迹中起着重要作用。在这项研究中，首先发展了DNA复制分析的基本技术。利用新的和以前的技术，研究了染色质的表观遗传修饰对DNA复制的影响。主要研究结果如下：1.采用低渗位移法将修饰的核苷酸引入细胞内，用于DNA复制分析。与载珠方法不同的是，这种方法不仅可以用于贴壁细胞，也可以用于浮动细胞。2.建立了DNA分子在显微镜盖玻片上精确、均匀延伸的动态分子梳理方法，并将其应用于DNA复制分析。还建立了一种从单个细胞制备半延伸DNA纤维的方法。使用此方法，…之间的异步复制3DNA纤维的直接观察表明，在S早期-中期，当复制叉穿过R/G染色体带边界和着丝粒周围异染色质时，复制叉移动的速率典型地降低。研究还表明，组蛋白超乙酰化与着丝粒异染色质区域的染色质结构、复制时间(起始激发)和分叉进程的变化密切相关。S阶段的检查点因子被认为在核DNA被破坏时负责抑制起始激发和复制叉子的稳定。4.核DNA通常在复制前和复制后分别与核基质结合和释放，尽管MAR区域一直通过S阶段与其结合。5.通过印迹结构域的复制时序分析和ES细胞延伸的复制DNA纤维的可视化等，促进了DNA复制在表观遗传学中在哺乳动物发育中的作用的基础研究。较少

Epigenetic modifications of chromatin play important roles in genomic imprinting. In this research, basic techniques for the analysis of DNA replication were developed first. Using new and previous techniques, effects of epigenetic modifications of chromatin on DNA replication were examined. The results obtained are listed as follows :1.A hypotonic shift method to introduce modified nucleotides into cells was applied for the analysis of DNA replication. In contrast to the beads-loading method, this can be used not only for adherent cells but for floating cells. Other advantages including efficient and homogeneous incorporation of modified nucleotides were also demonstrated.2.A dynamic molecular combing method that extends DNA molecules precisely and uniformly on a microscope cover slip was established, and applied to analysis of DNA replication. A preparation method of semi-extended DNA fibers from individual cells was also developed. Using this method, asynchronous replication between … More parental alleles in imprinted regions was demonstrated.3.Direct visualization of DNA fibers showed that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. It was also shown that histone hyperacetylation is tightly associated with changes in chromatin structure, replication timing (origin firing) and fork progression of centromeric heterochromatin regions. S phase checkpoint factors were suggested to be responsible for repression of origin firing and stabilization of replication forks when nuclear DNAs were damaged.4.Nuclear DNAs generally bind and release to the nuclear matrix just before and after their replication, respectively, although MAR regions consistently bind to it through S phase.5.Basic researches to examine roles of DNA replication in epigenetics in mammalian development were promoted through replication timing analysis of imprinted domains, visualization of replicating DNA fibers extended from ES cells, and so on. Less