DNA Replication Initiation Sites in Mammalian Cells

描述（由申请人提供）：几年来，我们一直在研究特定DNA序列中的复制起始。我们的单分子分析复制DNA（SMARD）的发展使我们能够研究包含高达1 Mb的基因组位点的片段的复制。在本提案中，我们将使用这种强大的方法来检查三个模型系统中的复制：Igh基因座，人类和小鼠端粒，以及含有三重重复序列的脆性X智力迟钝（FMR 1）基因座。我们已经证明，在非B细胞中，在IgH恒定区基因座的一个区域中不存在起源，但在非B细胞中，IgH恒定区基因座的一个区域被激活。 pro-B细胞中的优选位点。我们将主要使用IgH-C基因座作为模型来检查对复制应激的反应，包括潜在起源的激活。我们将通过ChIP确定IgH-C基因座中的复制前复合物（pre-RC）定位和核小体组织。比较SMARD起源定位与前RC占用和核小体景观将提供洞察潜在的起源激活的机制。在第二个模型中，我们在SMARD中取得了重要的技术进步，使我们能够研究特定端粒位点的复制，并获得了端粒复制程序的详细信息。一个关键的发现是，人类和小鼠的端粒似乎是通过染色体特异性复制程序而不是通用程序复制的。我们将使用SMARD来确定这些程序如何受到复制压力的影响，以及复制的哪些方面受到端粒的挑战。我们还将检查特定端粒的端粒功能障碍。这些分析将揭示端粒复制程序的灵活性，包括激活潜在起源的能力，并确定特定的复制程序是否使端粒更容易受到压力和功能障碍的影响。我们也有证据表明，BLM解旋酶直接有助于有效的端粒复制。我们将使用SMARD来确定BLM如何促进端粒复制，以及端粒是否比内部基因组区域更依赖于BLM。我们还将确定端粒复制中BLM和其他解旋酶之间的功能重叠。在第三个模型中，我们将研究三核苷酸重复序列（TNR）扩增，这与包括脆性X综合征（FXS）在内的30多种遗传性疾病有关。已经产生了许多模型系统来研究复制在TNR扩增中的作用，但是缺乏在内源性人类基因座的扩增模型。使用SMARD，我们现在能够比较人类细胞中内源性FMR 1基因座（FXS中受影响的基因座）中复制起始位点在TNR扩增中的作用，包括来自具有生育FXS男性风险的女性的细胞系，其具有和不具有TNR扩增，以阐明起始位点选择对TNR不稳定性的作用。我们还将确定CTCF结合FMR 1复制程序的贡献。基于我们的初步数据，我们预计这些拟议的研究将大大增加我们对端粒和TNR复制的理解，并使我们能够建立新的模式，复制起点选择在健康和疾病中的作用。

DESCRIPTION (provided by applicant): For several years we have studied replication initiation in specific DNA sequences. Our development of Single Molecule Analysis of Replicated DNA (SMARD) has allowed us to study the replication of segments encompassing up to 1 Mb of a genomic locus. In the present proposal we will use this powerful approach to examine replication in three model systems: the Igh locus, human and mouse telomeres, and the triplet repeat- containing Fragile X Mental Retardation (FMR1) locus. We have shown that origins are absent in a region of the IgH-constant region locus in non-B cells but are activated at preferred sites in pro-B cells. We will use Igh- C locus primarily as a model to examine responses to replication stress, including activation of latent origins. We will determine pre-replication complex (pre-RC) localization and nucleosome organization in the Igh-C locus by ChIP. Comparison of SMARD origin mapping with pre-RC occupancy and nucleosome landscape will provide insights into the mechanisms of latent origin activation. In the second model we have made essential technical advances in SMARD that allow us to study replication in specific telomere loci and have obtained detailed information on telomere replication programs. A key finding is that human and mouse telomeres appear to be replicated by chromosome-specific replication programs rather than a universal program. We will use SMARD to determine how these programs are affected by replication stress and what aspects of replication are challenged by telomeres. We will also examine telomere dysfunction at specific telomeres. These analyses will reveal the flexibility of telomere replication programs, including the ability to activate latent origins, and establish if specific replication programs render telomeres more susceptible to stress and dysfunction. We also have evidence to suggest that BLM helicase directly contributes to efficient telomere replication. We will use SMARD to determine how BLM facilitates telomere replication and if telomeres are more reliant on BLM than internal genomic regions. We will also determine the functional overlap between BLM and other helicases in telomere replication. In the third model we will study trinucleotide repeat (TNR) expansion, which is associated with more than 30 inherited diseases including Fragile X syndrome (FXS). Many model systems have been generated to study the role of replication in TNR expansion, but models of expansion at endogenous human loci are lacking. Using SMARD we are now able to compare the role of replication initiation sites in TNR expansion in the endogenous FMR1 locus (the affected locus in FXS) in human cells, including cell lines from women at risk for giving birth to FXS males, that have and do not have TNR expansion to elucidate the role of origin site selection on TNR instability. We will also determine the contribution of CTCF binding to FMR1 replication programs. Based on our preliminary data, we expect these proposed studies will both greatly increase our understanding of telomere and TNR replication and allow us to establish new paradigms for the role replication origin selection in health and disease.