DNA Replication initiation Sites in Mammalian Cells

描述（由申请人提供）：我们的长期目标是了解哺乳动物细胞中DNA复制起始决定因素的控制。我们为此目的开发的系统依赖于小鼠免疫球蛋白重链基因（Igh）位点的复制起始位点的鉴定。我们已经确定，在B谱系发育的早期和晚期，这个位点的复制时间在细胞之间是完全不同的。在发育早期的细胞中，整个Igh基因座在S期早期复制，而在发育后期，部分基因座在S期后期复制。我们推测复制起始位点的数量和位置在B细胞发育过程中发生了变化。我们现在建议在整个分化过程中跟踪B系细胞的这些变化。我们还将研究组蛋白修饰和新复制起始位点激活之间的关系。

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.