Preferential re-replication of Drosophila heterochromatin in the absence of geminin.
Preferential re-replication of Drosophila heterochromatin in the absence of geminin.
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DOI：
10.1371/journal.pgen.1001112
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发表时间：
2010-09-09
期刊：
影响因子：
4.5
通讯作者：
MacAlpine DM
中科院分区：
文献类型：
--
作者：
Ding Q;MacAlpine DM
为了确保基因组的完整性，基因组必须在每个细胞周期精确复制一次。复制许可机制的破坏可能导致再复制和基因组不稳定。Cdt1在果蝇中也被称为double - parking (Dup)，是pre- replication complex （pre-RC）组装的关键调节因子，其活性受到多种机制的严格限制，包括Cul4-Ddb1介导的蛋白水解和双胞蛋白的抑制。我们分析了通过RNAi耗尽双胞蛋白来失调复制许可机制的基因组后果。我们发现并不是所有的复制起源都对双染色体耗尽敏感，而且异染色质序列在没有许可机制的情况下优先被重新复制。异染色质起源复制的优先再激活是出乎意料的，因为这些通常是在正常细胞周期中最后被复制的序列。我们发现异染色质的再复制不是在pre-RC激活的水平上调控的，而是通过pre-RC的形成来调控的。与在G1期整个基因组中发生的pre-RC的全局组装不同，在没有双联蛋白的情况下，有限的pre-RC组装被细胞周期蛋白A-CDK活性升高限制在异染色质上。这些结果表明，G1外存在染色质和细胞周期特异性调控pre-RC的重组。如果细胞不能完全复制基因组，或者在一个细胞周期中复制基因组的某些区域不止一次，可能会发生灾难性的后果。细胞必须协调数千个DNA复制起始位点（起点），以确保整个基因组被复制，并且在细胞周期中没有复制起点被激活超过一次。细胞通过将复制起点的选择和激活限制在细胞周期的离散阶段来完成这种协调。起始位点只能在G1期被选择或“许可”用于DNA复制，同样，它们只能在S期被激活用于DNA复制的起始。调控这一“许可”过程的机制的破坏已被证明会在各种真核系统中导致广泛的再复制、基因组不稳定和肿瘤发生。在这里，我们在果蝇中使用基因组方法来确定在没有复制许可控制的情况下，哪些复制起源容易受到DNA复制重新启动的影响。出乎意料的是，我们发现异染色质中的序列，被认为只包含低效率的复制起源，被优先重新复制。这些结果提供了如何在不同的染色质环境中选择和调节复制起源以维持基因组稳定性的见解。
To ensure genomic integrity, the genome must be duplicated exactly once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. Cdt1, also known as Double-parked (Dup) in Drosophila, is a key regulator of the assembly of the pre-replicative complex (pre-RC) and its activity is strictly limited to G1 by multiple mechanisms including Cul4-Ddb1 mediated proteolysis and inhibition by geminin. We assayed the genomic consequences of disregulating the replication licensing mechanisms by RNAi depletion of geminin. We found that not all origins of replication were sensitive to geminin depletion and that heterochromatic sequences were preferentially re-replicated in the absence of licensing mechanisms. The preferential re-activation of heterochromatic origins of replication was unexpected because these are typically the last sequences to be duplicated in a normal cell cycle. We found that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather by the formation of the pre-RC. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin by elevated cyclin A-CDK activity. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1. Catastrophic consequences may occur if the cell fails to either completely copy the genome or if it duplicates some regions of the genome more than once in a cell cycle. The cell must coordinate thousands of DNA replication start sites (origins) to ensure that the entire genome is copied and that no replication origin is activated more than once in a cell cycle. The cell accomplishes this coordination by confining the selection and activation of replication origins to discrete phases of the cell cycle. Start sites can only be selected or ‘licensed’ for DNA replication in G1 and similarly, they can only be activated for the initiation of DNA replication in S phase. Disruption of the mechanisms that regulate this ‘licensing’ process have been shown to result in extensive re-replication, genomic instability and tumorigenesis in a variety of eukaryotic systems. Here we use genomic approaches in Drosophila to identify which origins of replication are susceptible to re-initiation of DNA replication in the absence of replication licensing controls. Unexpectedly, we find that sequences in the heterochromatin, which were thought to contain only inefficient origins of replication, are preferentially re-replicated. These results provide insights into how origins of replication are selected and regulated in distinct chromatin environments to maintain genomic stability.
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