INITIATION OF DNA REPLICATION IN MAMMALIAN CHROMOSOMES

为了描述控制启动的分子事件， DNA合成在哺乳动物染色体，我们已经研究了启动和 扩增的二氢叶酸还原酶内DNA复制的进展 甲氨蝶呤抗性中国仓鼠细胞株的DHFR结构域， CHOC 400. 使用在细胞周期中合成的新生DNA的杂交研究 在完整或透化的细胞中S期的开始已经表明复制 的扩增的DHFR结构域起始于4.3kb Xba I片段内 其定位在扩增的DHFR基因下游14 kb处。 我们建议 通过映射完成起始区域的物理表征， 对DHFR复制起点周围的11 kb DNA进行测序。 我们 将同时进行链特异性杂交研究， 冈崎片段，以精确定位内的起始位点 DHFR起始片段。 此外，我们还将增加 模板序列与交联剂和核酸酶在透化处理中的相互作用 G1/S细胞，以确定限制体外复制的条件 到初始化事件。 这些本地化研究将与 电子显微镜检查内的起始事件 扩增域 初步的电磁观测显示 二级结构，可能代表新的中间体， 启动DNA合成。 为了鉴定和分离蛋白质 参与DNA合成起始的因子，特异性染色质 起源区域内的配置将首先由 核酸酶消化研究;与起始相关的染色质复合物 DNA合成的平行复制研究将确定在 透化细胞 然后我们将使用改良的Western blot， 竞争和DNA纤维素亲和层析技术， 鉴定纯化的核提取物中以高亲和力结合的蛋白质 和对DHFR起源区域的特异性。 最后，我们将使用 用于蛋白结合研究的核蛋白提取物， 互补实验，以努力开发一种体外测定， 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.