DNA Replication initiation Sites in Mammalian Cells

描述(申请人提供)：我们的长期目标是了解在哺乳动物细胞中启动DNA复制的决定因素的控制。我们为此开发的系统依赖于鉴定小鼠免疫球蛋白重链基因(IgH)基因座上的复制起始点。我们已经证实，在B细胞系发育的早期和晚期，该基因座上的复制时间是完全不同的。在发育早期的细胞中，整个IgH基因座在S期早期复制，而在后期，部分基因座在S期复制。我们推测，在B细胞发育过程中，复制起始点的数量和位置会发生变化。我们现在建议跟踪B系细胞在分化过程中的这些变化。我们还将研究组蛋白修饰和新复制起始点激活之间的关系。 我们将依赖于我们开发的新方法，如我们先前应用的目标所述，用于研究哺乳动物细胞中单拷贝基因位点的DNA复制；我们称之为复制DNA的单分子分析(SMARD)。这种方法最近使我们能够分析哺乳动物细胞单拷贝免疫球蛋白基因座上的复制起始点。它进一步为我们提供了一种手段，通过B细胞发育的所有阶段，在细胞系和原代细胞中跟踪IgH基因位点的复制。后者以前并不适合研究，因为传统的电泳法在每个发育阶段都需要大量的细胞。正常细胞和恶性细胞都将被研究。我们的研究将确定复制起始点的使用是否确实随着发育而改变，最终将有可能解决IgH基因座上的复制分叉方向是否控制一些B细胞特有的过程，如VDJ重组、同型转换和体细胞超突变。我们将使用SMARD来定位和表征代表连续分化阶段的B细胞系和原代细胞中受发育调控的复制起始点。我们将研究整个免疫球蛋白基因座的DNA复制，以确定在PRO和Pre B细胞中活跃的复制起始点，而在代表B细胞发育后期的细胞中沉默的复制起始点。我们将研究在B细胞系和原代细胞中，组蛋白在免疫球蛋白基因中被激活和沉默的发育调节起始点的乙酰化状态。最后，我们将在ES细胞中使用基因打靶来确定转录的影响，可能是通过修改染色质结构，对原始B系细胞复制起点的激活。

DESCRIPTION (provided by applicant): Our long term goal is to understand the control of the determinants of initiation of DNA replication in mammalian cells. The system we have developed for this purpose rests on the identification of the replication initiation sites in the mouse immunoglobulin heavy chain gene (Igh) locus. We have established that the timing of replication at this locus differs radically between cells in the early and late stages of B lineage development. In cells in the early stages of development the entire Igh locus replicates early in the S phase, while in the late stages part of the locus replicates later in S phase. We inferred that the number and location of replication initiation sites changes during B cell development. We propose now to track these changes in cells of the B lineage throughout the course of differentiation. We will also examine the relationship between histone modifications and the activation of new replication initiation sites. We will rely on the new approach that we have developed, as set out in the objectives of our previous application, for the study of DNA replication in single-copy gene loci in mammalian cells; we have termed this Single Molecule Analysis of Replicated DNA (SMARD). This approach has recently enabled us to analyze the replication initiation sites in the single copy Igh locus of mammalian cells. It further affords us now a means of following, through all the stages of B cell development, the replication of the Igh locus in both cell lines and primary cells. The latter have not previously been amenable to study because of the large number of cells in each developmental stage demanded by the conventional, electrophoretic, method of analysis. Both normal and malignant cells will be studied. Our studies will establish whether indeed replication origin usage changes with development and ultimately will make it possible to resolve the question of whether the replication fork direction in the Igh locus governs some B cell specific processes such as VDJ recombination, isotype switching and somatic hypermutation. We will use SMARD to localize and characterize the developmentally regulated replication initiation sites in B cell lines and primary cells representing successive stages of differentiation. We will study DNA replication throughout the Igh locus in order to identify replication initiation sites that are active in pro and pre B cells and silenced in cells representing later stages of B cell development. We will study the acetylation status of histones at activated and silenced developmentally regulated initiation sites in the Igh locus in B cell lines and primary cells. Finally, we will use gene targeting in ES cells to determine the impact of transcription, possibly through the modification of chromatin structure, on the activation of replication origins in primary B lineage lineage cells.