Rad3 decorates critical chromosomal domains with gammaH2A to protect genome integrity during S-Phase in fission yeast.
Rad3 decorates critical chromosomal domains with gammaH2A to protect genome integrity during S-Phase in fission yeast.
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DOI：
10.1371/journal.pgen.1001032
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发表时间：
2010-07-22
期刊：
影响因子：
4.5
通讯作者：
Russell P
中科院分区：
文献类型：
--
作者：
Rozenzhak S;Mejía-Ramírez E;Williams JS;Schaffer L;Hammond JA;Head SR;Russell P
裂糖酵母Rad3检查点激酶及其人类同源物ATR对于在基因毒素损伤DNA或阻止复制叉的细胞中维持基因组完整性至关重要。Rad3和ATR在未受干扰的生长过程中也起作用，尽管触发它们激活的事件及其关键功能在很大程度上是未知的。在这里，我们使用ChIP-on-chip分析来绘制由磷酸化组蛋白H2A （γH2A）修饰的基因组位点，这是一种Rad3底物，为Crb2和Brc1 DNA修复/检查点蛋白建立了基于染色质的募集平台。出乎意料的是，γ - h2a在s期标记了多种基因组特征，包括自然复制叉屏障和叉断裂位点、反转录转座子、着丝粒和端粒中的异染色质以及核糖体RNA （rDNA）重复序列。着丝粒和端粒的γ - h2a形成与Clr4组蛋白甲基转移酶建立异染色质有关。我们发现γ - h2a结构域招募Brc1，这是一种参与修复受损复制分叉的因子。Brc1 c端BRCT结构域与γH2A的结合在Rqh1Sgs1缺失的情况下是至关重要的，Rqh1Sgs1是rDNA维持所需的RecQ DNA解旋酶，其人类同源物在以癌症易感或加速衰老为特征的Werner， Bloom和rothmond - thomson综合征患者中发生突变。我们得出结论，Rad3磷酸化组蛋白H2A，将Brc1动员到s期的关键基因组区域，并且该途径与Rqh1 DNA解旋酶平行发挥作用，以维持基因组的完整性。真核生物基因组的大小范围从~ 107到~ 1011个碱基对，每个细胞周期都以几乎绝对的保真度进行复制。尽管复制分叉经常出现停滞或崩溃，但这一惊人的壮举还是发生了。检查点激酶ATR被复制叉停止激活，并磷酸化受损DNA周围核小体中的组蛋白H2A。由于触发ATR激活的基因组区域在很大程度上是未知的，我们使用全基因组微阵列来绘制裂变酵母DNA复制过程中富含phospho-H2A的染色体结构域。该分析确定了特定的位点，包括核糖体DNA重复序列中的自然复制叉屏障，反转录转座子元件，最令人惊讶的是，所有的异染色质区域。Phospho-H2A结合基因组维持蛋白Brc1，我们的遗传学研究表明，在Rqh1缺失的情况下，这一分子途径变得至关重要，Rqh1是一种与癌症易感相关的保守DNA解旋酶。由于分裂酵母和人类基因组有许多相似之处，我们的研究揭示了可能类似地触发人类细胞中ATR激活的基因组标记，并表明phospho-H2A和Brc1是在DNA复制过程中维持基因组完整性的网络的关键部分。
Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (γH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, γH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. γH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that γH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to γH2A is crucial in the absence of Rqh1Sgs1, a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund–Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity. Eukaryotic genomes, which range in size from ∼107 to ∼1011 base pairs, are replicated with nearly absolute fidelity every cell cycle. This amazing feat happens despite the frequent stalling or collapse of replication forks. The checkpoint kinase ATR is activated by replication fork stalling and phosphorylates histone H2A in nucleosomes surrounding damaged DNA. As the genomic regions triggering ATR activation are largely unknown, we used a whole-genome microarray to map chromosomal domains enriched with phospho-H2A during DNA replication in fission yeast. This analysis identified specific sites, including natural replication fork barriers in ribosomal DNA repeats, retrotransposon elements, and most surprisingly, all heterochromatin regions. Phospho-H2A binds the genome maintenance protein Brc1, and our genetic studies reveal that this molecular pathway becomes crucial in the absence of Rqh1, a conserved DNA helicase that is linked to cancer predisposition. As the fission yeast and human genomes share many similarities, our study reveals genomic landmarks that could similarly trigger ATR activation in human cells and shows that phospho-H2A and Brc1 are a critical part of the network that maintains genome integrity during DNA replication.
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