REGULATION OF DNA SYNTHESIS IN MAMMALIAN CELLS

DNA合成的启动是哺乳动物体内的一个关键控制点 细胞周期。Okazaki片段的链特异性杂交研究 已经将双向DNA复制(OBR)的起始点映射到950 bp 中国仓鼠二氢叶酸还原酶(DHFR)基因的3‘端。这个 DHFR起始区包含稳定弯曲的DNA片段，可与 多种细胞因子，包括新的DNA结合蛋白RIP60， 这与依赖于ATP的DNA解旋酶RIP100相互作用。这项建议 旨在了解Rip60/RIP100和其他 细胞因子在调节DHFR中DNA合成的启动中发挥作用 起源。RIP60和RIP100将通过建立的 协议；纯化的蛋白质将用于蛋白质测序、生化 分析了Rip60/RIP100的功能，并制作了特定的 抗体。蛋白质序列数据将用于识别cdna克隆 RIP60和RIP100。Rip60和RIP100的cDNA克隆和抗体将 用于研究这些因子的表达和细胞定位 在整个细胞周期中。Rip60和Rip60之间关联的性质 RIP100，在存在和不存在特定和非特定DNA的情况下 序列，将通过扫描透射电子显微镜进行研究， 凝胶过滤和沉淀技术。解旋酶活性的测定 将研究RIP100的ATPase活性、解离能力 带有和不带有Rip60结合位点的dsDNA，以及解开ds的能力 含有起始序列的质粒。单链DNA的作用 这些反应上的结合蛋白(如SSP、基因32和RF-A)将是 已评估。Rip60等蛋白结合部位在体内的占据 将作为细胞周期的函数进行检查。一种稳定的转染法 在整合到新的基础上，将使用检测来研究起源功能 在基因组中的位置。重点将放在研究角色上 在起源功能上的弯曲DNA。DNA序列的鉴定 起源功能所必需的，以及涉及到的蛋白质的纯化 原点激活，将提供有关 细胞生长、分化和分化过程中的增殖调控 疾病。

Initiation of DNA synthesis is a critical control point in the mammalian cell cycle. Strand-specific hybridization studies with Okazaki fragments have mapped an origin of bidirectional DNA replication (OBR) to a 950 bp region 3' to the Chinese hamster dihydrofolate reductase (dhfr) gene. The dhfr origin region contains a fragment of stably bent DNA that binds multiple cellular factors, including the novel DNA binding protein, RIP60, that copurifies with the ATP-dependent DNA helicase, RIP100. This proposal is directed toward understanding the role that RIP60/RIP100 and other cellular factors play in regulating initiation of DNA synthesis at the dhfr origin. RIP60 and RIP100 will be purified in quantity by an established protocol; purified protein will be used for protein sequencing, biochemical analysis of RIP60/RIP100 function, and the production of specific antibodies. Protein sequence data will be used to identify cDNA clones for RIP60 and RIP100. cDNA clones and antibodies to RIP60 and RIP100 will be used to study the expression and cellular location of these factors throughout the cell cycle. The nature of the association between RIP60 and RIP100, in the presence and absence of specific and nonspecific DNA sequences, will be studied by scanning transmission electron microscopy, gel filtration, and sedimentation techniques. The helicase activity of RIP100 will be studied in regard to ATPase activity, ability to unwind dsDNA with and without RIP60 binding sites, and ability to unwind ds plasmids containing origin sequences. The effect of single-stranded DNA binding proteins (e.g. SSP, gene 32, and RF-A) on these reactions will be evaluated. The occupation of RIP60 and other protein binding sites in vivo will be examined as a function of the cell cycle. A stable transfection assay will be used to study origin function after integration into new positions in the genome. An emphasis will be placed on studying the role of bent DNA in origin function. The identification of DNA sequences required for origin function, and the purification of proteins involved in origin activation, will provide fundamental information concerning the regulation of cellular proliferation during growth, differentiation, and disease.