A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M.
A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M.
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DOI：
10.1371/journal.pgen.1006277
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发表时间：
2016-08
期刊：
影响因子：
4.5
通讯作者：
Schwacha A
中科院分区：
文献类型：
--
作者：
Vijayraghavan S;Tsai FL;Schwacha A
Mcm 2 -7复合物是真核生物复制解旋酶的催化核心。在这里，我们确定了一个新的角色，这个复杂的维持基因组的完整性。使用遗传学和细胞学方法，我们发现一个特定的mcm等位基因（mcm 2DENQ）导致基因组不稳定性升高，与γ H2 AX和Rad 52的许多DNA损伤相关病灶的出现相关。我们进一步发现，这种基因组不稳定性的触发事件是RNA：DNA杂交体水平升高和DNA拓扑状态改变，因为RNaseH（一种特异性降解RNA：DNA杂交体中RNA的酶）或拓扑异构酶1（一种减轻DNA超螺旋的酶）的过度表达可以抑制mcm 2DENQ DNA损伤表型。此外，观察到的DNA损伤有几个额外的不寻常的属性，在DNA损伤灶只出现在S期后，在G2/M，并依赖于进展到中期。此外，我们表明，由此产生的DNA损伤不是由于自发的S-相叉崩溃。总的来说，这些不寻常的mcm 2DERQ表型与以前研究的检查点传感器激酶ATR/MEC 1的一个特殊等位基因的表型明显相似，这表明Mcm 2 -7和ATR在未受挑战的生长过程中可能存在相互调节作用。由于RNA：DNA杂交主要是由于转录扰动，我们认为，监视介导的调制的Mcm 2 -7活动在防止复制叉和转录复合物之间的灾难性冲突中起着重要作用。这些影响之间的可能关系和最近发现的作用，Mcm 2 -7在DNA复制检查点诱导HU治疗进行了讨论。DNA复制的精确调控是避免基因组不稳定性所必需的，基因组不稳定性是遗传信息的有害改变，是癌症等疾病的标志。然而，复制不仅仅是遗传密码的简单复制。复制机制还有一个额外的问题--它必须以某种方式调节延伸，以成功地避开与障碍物或其他正在进行的染色质依赖性过程（例如，转录）。这种冲突会导致基因组的不稳定性，人们对它们通常是如何避免的知之甚少。使用芽殖酵母作为我们的模型系统，我们表明，这种损害的避免可能需要直接参与的Mcm 2 -7复制解旋酶，分子马达，在复制过程中解开DNA。Mcm复合物内的关键活性位点的改变导致基因组不稳定性的显著水平。令人惊讶的是，我们的证据表明，通常与转录相关的问题（例如，RNA：DNA杂交体）是这种损伤的最终来源。这一结果意味着一个新的和积极的作用，复制解旋酶在调节延长，成功地导航选择基因组障碍，并奠定了基础，以进一步探索改变DNA复制的基因组稳定性的背景下的分子原因和后果。
The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. Here, we identify a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, we find that a specific mcm allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX and Rad52. We further find that the triggering events for this genome instability are elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, we show that the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1, suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, we suggest that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed. The precise regulation of DNA replication is necessary to avoid genome instability, the deleterious alteration of genetic information that is a hallmark of diseases like cancer. However, replication is more than just a simple duplication of the genetic code. The replication machinery has an additional problem–it must somehow regulate elongation to successfully elude potentially destructive conflicts with obstacles or other ongoing chromatin-dependent processes (e.g., transcription). Such conflicts generate genome instability, and how they are normally avoided is poorly understood. Using budding yeast as our model system, we show that such damage-avoidance likely requires direct involvement of the Mcm2-7 replicative helicase, the molecular motor that unwinds DNA during replication. Alteration of a key active site within the Mcm complex results in substantial levels of genome instability. Surprisingly, our evidence indicates that problems normally associated with transcription (e.g., RNA:DNA hybrids) are the ultimate source of this damage. This result implies a novel and active role for the replicative helicase in regulating elongation to successfully navigate select genomic barriers, and lays the groundwork to further explore the molecular causes and consequences of altering DNA replication in the context of genome stability.