INITIATION OF DNA REPLICATION IN MAMMALIAN CHROMOSOMES

为了描绘控制分子事件启动的分子事件 在哺乳动物染色体中的DNA合成，我们已经研究了启动和 扩增的二氢叶酸还原酶内DNA复制的研究进展 抗甲氨蝶呤中国仓鼠细胞株的(DHFR)结构域， 乔克400。杂交研究中合成的新生DNA S期在整个或通透性细胞中的开始表明复制 在4.3kb的Xba I片段中起始 该基因定位于扩增的dhfr基因下游14kb。我们建议 通过作图和映射来完成起始区的物理表征 对dhfr复制起点周围的11kb DNA进行测序。我们 将同时进行链特异性杂交研究 Okazaki碎片以便精确定位其中的起始点 Dhfr起源片段。此外，我们还将介绍 渗透剂中交联剂和核酸酶的模板序列 G1/S细胞体外复制限制条件的确定 到入会仪式。这些本地化研究将与 用电子显微镜观察核反应中的起爆事件 扩增结构域。初步的EM观察发现了异常情况 可能代表新中间体的二级结构 DNA合成的启动。为了鉴定和分离蛋白质 参与启动DNA合成的因素，特异性染色质 源区域内的配置将首先由 核酸酶消化研究；与起始有关的染色质复合体 的DNA合成将通过平行复制研究在 通透性细胞。然后我们将使用改良的Western印迹，条带 竞争和DNA纤维素亲和层析技术 鉴定纯化的核提取液中与高亲和力结合的蛋白质 和DHFR起始区的特异性。最后，我们将使用 用于蛋白质结合研究的核蛋白提取液 互补实验，以期建立一种新的体外检测方法 在纯化的细胞核中启动DNA合成。

In an effort to delineate the molecular events that control initiation of DNA synthesis in mammalian chromosomes, we have studied initiation and progression of DNA replication within the amplified dihydrofolate reductase (DHFR) domains of the methotrexate-resistant Chinese hamster cell strain, CHOC 400. Hybridization studies using nascent DNA synthesized during the onset of S phase in whole or permeabilized cells has shown that replication of the amplified DHFR domains is initiated within a 4.3 kb Xba I fragment that maps 14 kb downstream from the amplified DHFR gene. We propose to complete physical characterization of the initiation region by mapping and sequencing 11 kb of DNA surrounding the DHFR origin of replication. We will simultaneously conduct strand specific hybridization studies with Okazaki fragments in order to precisely locate the initiation sites within the DHFR origin fragment. In addition, we will intrate the sensitivity of template sequences to crosslinking agents and nucleases in permeabilized G1/S cells in order to define conditions for limiting replication in vitro to initiation events. These localization studies will be correlated with an electron microscopic examination of initiation events within the amplified domain. Preliminary EM observations have revealed unusual secondary structures that likely represent novel intermediates in initiation of DNA synthesis. In order to identify and isolate protein factors involved in initiation of DNA synthesis, specific chromatin configurations within the origin region will be first delineated by nuclease digestion studies; those chromatin complexes related to initiation of DNA synthesis will be identified by parallel replication studies in permeabilized cells. We will then use modified Western blot, band competition, and DNA cellulose affinity chromatography techniques to identify proteins in purified nuclear extracts that bind with high affinity and specificity to the DHFR origin region. Finally, we will use the nuclear protein extracts prepared for protein binding studies in complementation experiments in an effort to develop an in vitro assay for initiation of DNA synthesis in purified nuclei.