CHROMATIN STRUCTURE AND INITIATION OF DNA REPLICATION

我们实验室的长期目标是表征顺式和反式 调控哺乳动物DNA复制启动的作用因子 染色体。已经设计了几种起源映射技术，这些技术 提示DNA复制起始于dhfr之间55kbp的区域 2BE2121基因在中国仓鼠卵巢细胞中表达。初步结果来自 我们的实验室表明，就像在酵母中一样，细胞周期调节的超敏部位 并且核酸酶抗性结构域出现在首选的DHFR基因座之一上 起始点，或贝塔。因为兽人情结被牵连到 在这些染色质结构的形成中扮演着随意的角色，并且作为 这些染色质结构被认为是一种调节 关于DNA复制的启动，已经产生了两个假设：(I) CHO细胞中Ori-Beta区染色质的组织与起源有关 功能，以及(Ii)染色质结构是由局部结合引起的 兽人复合体。这些假设将通过以下方式得到解决 具体目标：1)确定染色质修饰是否相互关联 在体内具有DHFR来源的活性。连接介导的聚合酶链式反应技术将 用于识别半合子CHO中染色质修饰的区域 细胞，以使启动阳性CHO细胞中的染色质状态 与起始阴性CHO细胞中的染色质状态相比。这 将证明染色质构型是(或不是) 与功能起源相关的。2)确定分发/绑定 ORC复合体在DHFR基因座内的位置，特别是在Ori- 贝塔。DNA相互作用因子交联的新方法 将被用来确定ORC在DHRF起源基因座中的结合位置。 然后将进行实验，以解决这些之间的关联 ORC：DNA与体内起源功能的相互作用。对这些问题的调查 提问将有助于更好地理解 并将提供对哺乳动物DNA复制的更清晰的理解 调控G1/S转换的机制。

The long-term goal of our laboratory is to characterize cis- and trans- acting factors that regulate initiation of DNA replication in mammalian chromosomes. Several origin mapping techniques have been devised which suggest that DNA replication initiates in a 55kbp region between the DHFR and 2BE2121 genes in Chines hamster ovary cells. Preliminary results from our lab show that, as in yeast, a cell cycle-regulated hypersensitive site and a nuclease-resistant domain occur over one of the DHFR locus preferred initiation sites, ori-Beta. As the ORC complex has been implicated in playing a casual role in formation of these chromatin structures, and as these chromatin structures have been implicated in a regulatory role in initiation of DNA replication, two hypotheses have been generated: (i) the organization of chromatin at ori-Beta in CHO cells is involved in origin function, and (ii) the chromatin structures are caused by locally-bound ORC complex(es). These hypothesis will be addressed by the following Specific Aims: 1) Determine whether the chromatin modifications correlate with DHFR origin activity in vivo. Ligation-mediated PCR techniques will be used to identify regions of chromatin modification in hemizygous CHO cells so that the chromatin state in initiation-positive CHO cells can be compared to the chromatin state in initiation-negative CHO cells. This will demonstrate that the chromatin configuration is (or is not) associated with a functional origin. 2) Determine the distribution/binding sites of ORC complex(es) within the DHFR locus and specifically at ori- Beta. Novel approaches involving cross-linking of DNA-interactive factors will be used to determine where ORC binds within the DHRF origin locus. Experiments will then be performed addressing the association of these ORC:DNA interactions with origin function in vivo. Investigation of these questions will allow for a better understanding of the initiation of mammalian DNA replication and will provide a more clear understanding of the mechanisms regulating the G1/S transition.