DNA REPLICATION INITIATION SITES IN MAMMALIAN CELLS

我们将使用两个模型系统来研究人类细胞系中的DNA复制。 1.爱泼斯坦-巴尔病毒(EBV)核抗原I(EBNA-1)在 EB病毒重复序列家族复制屏障的形成 人类细胞系中的序列将使用体外复制进行研究 系统。我们将确定所需的最小重复次数 为了制造体外复制的屏障并确定EBNA-1 与重复序列家族的结合抑制T细胞的解旋酶活性 抗原。然后我们将确定细胞蛋白质是否也能产生 通过与EBV重复序列家族结合而形成的复制障碍。2.我们会 识别早期复制的rDNA基因簇中的复制起点 或者在S阶段的后期。 我们将确定复制的站点的位置 免疫球蛋白重链多基因家族启动。这将是 在表达这些基因中的一个或多个的细胞系中确定的 在转录沉默的细胞系中也是如此。我们将使用 进行缺失分析以确定对这些序列至关重要的序列 免疫球蛋白复制开始发挥作用。定向整合 由同源重组介导的将被用于修饰染色体 原产地区域。

We will use two model systems to study DNA replication in human cell lines. 1. The role of the Epstein-Barr virus (EBV) nuclear antigen I (EBNA-1) in the formation of a replication barrier at the EBV family of repeated sequences in human cell lines will be studied using an in vitro replication system. We will determine the minimum number of repeats that are required to produce a barrier to replication in vitro and determine whether EBNA-1 binding to the family of repeats inhibits helicase activity of the T antigen. We will then determine whether cellular proteins can also produce a replication barrier by binding to the EBV family of repeats. 2. We will identify replication origins in the rDNA gene cluster that replicate early or late during the S phase. We will determine the location of the sites at which the replication of the immunoglobulin heavy chain multigene family initiates. This will be determined in cell lines in which one or more of these genes are expressed and in cell lines in which they are transcriptionally silent. We will use deletion analysis to determine the sequences that are critical for these immunoglobulin replication origins to function. Targeted integration mediated by homologous recombination will be used to modify the chromosomal origin region.