REGULATION OF DNA SYNTHESIS IN MAMMALIAN CELLS

DNA合成的起始是哺乳动物中的关键控制点， 细胞周期 冈崎片段的链特异性杂交研究 已经将双向DNA复制（OBR）的起点定位到950 bp 中国仓鼠二氢叶酸还原酶（dhfr）基因的3 ′区。 的 dhfr起始区含有稳定弯曲的DNA片段， 多种细胞因子，包括新的DNA结合蛋白，RIP 60， 与依赖ATP的DNA解旋酶RIP 100共同纯化。 这项建议 旨在理解RIP 60/RIP 100和其他 细胞因子在调节dhfr的DNA合成起始中起作用 起源 RIP 60和RIP 100将通过已建立的 方案;纯化的蛋白质将用于蛋白质测序、生物化学 分析了RIP 60/RIP 100的功能，并制作了具体的 抗体的 蛋白质序列数据将用于鉴定cDNA克隆， RIP 60和RIP 100。 将使用cDNA克隆和针对RIP 60和RIP 100的抗体。 用于研究这些因子的表达和细胞定位 在整个细胞周期中。 RIP 60与 RIP 100，在存在和不存在特异性和非特异性DNA的情况下 序列，将通过扫描透射电子显微镜进行研究， 凝胶过滤和沉降技术。 解旋酶活性 将研究RIP 100的ATP酶活性、解链能力和细胞增殖能力。 具有和不具有RIP 60结合位点的dsDNA，以及解旋dsDNA的能力， 含有起始序列的质粒。 单链DNA的作用 这些反应中的结合蛋白（如SSP、基因32和RF-A）将被 评估。 RIP 60和其他蛋白结合位点在体内的占据 将作为细胞周期的函数进行检查。 稳定转染 分析将用于研究整合到新的 在基因组中的位置。 重点将放在研究的作用 弯曲的DNA的起源功能。 DNA序列的鉴定 所需的起源功能，并参与蛋白质的纯化 起源激活，将提供有关的基本信息， 在生长、分化和分化过程中调节细胞增殖， 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.