MODULATION OF ORI-BETA REPLICATION ORIGIN BY FISH OIL

描述（由申请人提供）： 众所周知，鱼油在人类和实验动物中具有显着的抗增殖和抗癌作用。在以前的研究中，我们已经确定了鱼油对S期进展的新作用。体内和体外研究均表明，补充鱼油后S期持续时间显著延长。这些效应与CHO细胞中位于二氢叶酸还原酶（DHFR）基因内的复制起点（ori-β）的活性变化相关。我们还表明，鱼油改变了细胞中的染色质结构，这种油对S期的影响得到了表达。S期延长和染色质结构的变化都通过向培养基中加入多胺前体腐胺来逆转。我们的假设是，鱼油通过改变复制起点的空间和/或时间组织来影响S期的持续时间。我们推测，鱼油改变S期和细胞周期进程的机制的阐明将取决于对哺乳动物细胞中特定DNA位点组织复制起点的分子机制的进一步理解。在这个小补助金申请中，我们建议启动研究，这将有助于未来对营养素在DNA复制中的作用的研究。最初，我们将使用详细的新生DNA图谱技术，在鱼油和PUFA处理后识别和表征ori-β、ori-β '和ori-γ的活性。这项研究将测试鱼油是否抑制ori-b并激活次级复制位点，或者这种治疗是否改变了复制起点的位置。我们还将进行可行性实验，以测试基因组DNA作图和交联/染色质免疫沉淀（CHIP）技术在DHFR基因座内复制蛋白/DNA相互作用研究中的适用性。 这个小应用的结果将帮助我们设计一个完整的研究项目，以确定鱼油和PUFA对DNA复制的分子靶点和作用机制。

DESCRIPTION (provided by applicant): Fish oil in known to have significant antiproliferative and anticancer effects in humans and experimental animals. In previous studies, we have identified a novel effect of fish oil on S-phase progression. Both in vivo and in vitro studies have shown that S-phase duration is significantly longer following fish oil supplementation. These effects are associated with a change in the activity at the replication origin (ori-beta) located within the dihydrofolate reductase (DHFR) gene in CHO cells. We have also shown that fish oil alters the chromatin structure in cells where the effect of this oil on S-phase is expressed. Both S-phase prolongation and the change in chromatin structure are reversed by addition of the polyamine precursor putrescine to the culture media. Our hypothesis is that fish oil affects the duration of S-phase by altering the spatial and/or temporal organization of replication origins. We postulate that elucidation of the mechanism(s) by which fish oil alter(s) S-phase and cell cycle progression will depend on further understanding of the molecular mechanisms that organize replication origins at specific DNA sites in mammalian cells. In this small grant application, we propose to initiate studies that will facilitate future research on the role of nutrients in DNA replication. Initially, we will identify and characterize the activity of ori-beta, ori-beta', and ori-gamma, following fish oil and PUFA treatment by use of a detailed nascent DNA mapping technique. This study will test whether fish oil suppresses ori-b and activates secondary replication sites, or whether this treatment alters the location of replication origins. We will also conduct feasibility experiments to test the applicability of genomic DNA mapping and cross-linking/chromatin immunoprecipitation (CHIP) techniques in studies of replication protein/DNA interactions within the DHFR gene locus. The results of this small application will help us design a full research project to determine the molecular targets and mechanism of action of fish oil and PUFA on DNA replication.