Crosslinks and replication: the show must go on.
Crosslinks and replication: the show must go on.
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交叉链接和复制：表演必须继续下去。
DOI：
10.1080/15384101.2015.1010971
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Sidorova,JuliaM
中科院分区：
文献类型：
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作者：
Sidorova,JuliaM
研究 DNA 复制对 DNA 损伤的反应对于我们了解癌症的起源和治疗至关重要。 DNA 损伤会减慢 S 期。根据对低等真核生物的研究，我们将这种减慢解释为损伤碰撞介导的复制叉停滞，从而触发 ATR-CHK1 检查点介导的起源激发（即复制启动）的全局抑制。实验表明，人类（和其他脊椎动物）细胞的情况更为复杂。其中一个几乎未被研究的方面是，如何对后生动物基因组复制的有序且保守的模式（称为 S 期程序）施加对起源放电的全局抑制。 S 阶段程序作为 Mb 大小的复制域的顺序激活来执行，每个复制域包含同时激发的多个起点（图 1，顶部）。 1 基因组可以比作一圈圣诞彩灯，它们大多是一个又一个循环地打开（移交或下一个排队事件），偶尔会有一些循环无序地打开（从头事件）。在任何一个时间参与复制的基因组分数和结构域数量在整个 S 期或多或少是恒定的，从而防止复制因子和前体的耗尽。 S 期程序的正确执行不仅可以确保基因组稳定性，还可以保留表观遗传景观，包括基因表达模式。但当 DNA 受损时，这种“灯光秀”会发生什么呢？ DNA 模板中的交联可以阻断试管中的 DNA 合成。在体内，这些病变极大地减慢了 S 期。然而，在人类细胞中，DNA 纤维分析（可以检测 DNA 损伤对个体复制叉进展、起点激发和复制叉终止的影响）显示交联对复制的影响很小甚至没有。最近，黄等人进行了一项优雅的研究。 2 建议作出解释。作者对 DNA 纤维中的复制轨迹和三恶英链间交联进行了可视化，并确定了一类主要事件，这些事件只能解释为复制叉“跳过”交联并继续向下游延伸。如果交联是可穿越的，那么整个 S 相会发生什么？我们分析了丝裂霉素 C (MMC) 交联剂对人体细胞 S 期的影响。 3 在细胞毒性剂量的 MMC 之前和之后的不同时间，我们使用多达 3 个胸苷类似物标记对原代人 S 期成纤维细胞和角质形成细胞中的 DNA 复制进行颜色编码和定量。通过流式细胞术和 DNA 纤维分析测量这种颜色编码的复制，使我们能够确定 MMC 处理的 S 期细胞在 DNA 损伤反应的同时保持了相当高水平的 DNA 合成。 MMC 复制前和后的分叉以相当的速率移动，然而，与 MMC 复制前和同期未处理的对照相比，MMC 复制后的分叉丰度在几个小时内减少了约 50%。如果有两个假设，则 MMC 后分叉丰度的适度减少可以解释 S 期的显着减慢
Research into responses of DNA replication to DNA damage is critical to our understanding of the genesis and treatment of cancer. DNA damage slows S phase. From studies in lower eukaryotes, we explain this slow-down as damage collision-mediated stalling of replication forks that triggers the ATR-CHK1 checkpoint-mediated global inhibition of origin firing (ie replication initiation). Experiments suggest that the story is more complex in human (and other vertebrate) cells. One virtually unstudied aspect of it is how the global inhibition of origin firing is imposed on the orderly and conserved mode of metazoan genomic replication known as the S phase program. The S phase program is executed as sequential activation of Mb-sized replication domains each of which contains multiple origins that fire simultaneously (Fig. 1, top). 1 The genome may be likened to loops of a garland of Christmas lights that turn on mostly one loop after another (hand-over or next–in-line events), with a few loops here and there turning on out of order (de novo events). The fraction of the genome and the number of domains engaged in replication at any one time is more or less constant throughout the bulk of S phase, preventing depletion of replication factors and precursors. Correct execution of the S phase program not only ensures genomic stability but may also preserve the epigenetic landscape, including gene expression patterns. But what happens to this “light show” when DNA is damaged? Crosslinks in the DNA template can block DNA synthesis in a test tube. In vivo, these lesions profoundly slow S phase. In human cells however, DNA fiber analyses (which can detect effects of DNA damage on progression of individual replication forks, origin firing, and fork termination) have been showing minor to no impact of crosslinks on replication. Recently, an elegant study by Huang et al. 2 suggested an explanation. The authors visualized both tracks of replication and trioxalen interstrand crosslinks in DNA fibers and identified a preponderant class of events that could only be interpreted as replication forks “jumping” over crosslinks and continuing downstream. If crosslinks are traversable, what happens to the S phase as a whole? We analyzed the effect of mitomycin C (MMC) crosslinker on S phase in human cells. 3 We used up to 3 thymidine analog labels to color-code and quantify DNA replication in primary human S phase fibroblasts and keratinocytes before and at various times after a cytotoxic dose of MMC. Measuring this color-coded replication by flow cytometry and DNA fiber analysis allowed us to determine that MMC-treated S phase cells maintained a reasonably high level of DNA synthesis concurrent with DNA damage response. Pre-and post MMC replication forks moved at comparable rates, however, for several hours the abundance of post-MMC forks was reduced by about 50% when compared to pre-MMC replication and contemporaneous untreated controls. This moderate reduction in post-MMC fork abundance can explain profound slowing of S phase if 2 assumptions are