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序列中的复制启动。我们开发的复制DNA单分子分析(SMARD)使我们能够研究包含高达1Mb的基因组位点的片段的复制。在目前的方案中，我们将使用这一强大的方法来检查三个模型系统的复制：IgH基因座、人和小鼠端粒，以及含有三联体重复的脆性X智力低下(FMR1)基因座。我们已经证明，在非B细胞中，IgH恒定区轨迹的某个区域没有起始点，但在 PRO-B细胞中的首选部位。我们将主要使用IGH-C基因座作为模型来研究对复制应激的反应，包括潜在起始点的激活。我们将通过芯片来确定复制前复合体(Pre-RC)在IgH-C基因座上的定位和核小体组织。将SMARD起源图谱与前RC占有率和核小体景观进行比较，将有助于深入了解潜在起源激活的机制。在第二个模型中，我们在SMARD方面取得了重要的技术进步，使我们能够研究特定端粒基因的复制，并获得了关于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特定于染色体的复制程序复制的，而不是一个通用的程序。我们将使用SMARD来确定这些程序如何受到复制压力的影响，以及端粒挑战复制的哪些方面。我们还将研究特定端粒上的端粒功能障碍。这些分析将揭示端粒复制程序的灵活性，包括激活潜在起始点的能力，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BLM。我们还将确定在端粒复制中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)的扩展，它与包括脆性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扩展中的作用，但缺乏内源性人类基因座的扩展模型。使用SMARD，我们现在能够比较内源性FMR1基因座(FXS中的受影响基因座)中复制起始位点在TNR扩张中的作用，包括来自有生育FXS男性风险的女性细胞系，这些细胞系有和没有TNR扩张，以阐明起始点选择在TNR不稳定性中的作用。我们还将确定CTCF结合对FMR1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极大地增加我们对端粒和TNR复制的理解，并使我们能够为复制来源选择在健康和疾病中的作用建立新的范式。

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.