Mitomycin C reduces abundance of replication forks but not rates of fork progression in primary and transformed human cells.
Mitomycin C reduces abundance of replication forks but not rates of fork progression in primary and transformed human cells.
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DOI：
10.18632/oncoscience.70
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Sidorova JM
中科院分区：
文献类型：
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作者：
Kehrli KR;Sidorova JM
DNA交联可以在体外阻断复制并在体内减缓S期进程。我们表征了丝裂霉素C交联剂对S期的整体影响以及对野生型和FANCD 2缺陷型人类细胞中的个体复制叉的影响。FANCD 2对交联修复至关重要，并且还涉及促进DNA复制。我们使用DNA纤维分析来证明MMC处理的细胞在S期期间复制叉的丰度持续降低，但进展率没有降低。FANCD 2缺陷并没有消除这种表型。EdU标记的DNA的免疫沉淀表明，在对MMC有反应的区域中，复制没有受到抑制，这一点以γ H2 AX的存在为标志，事实上，在野生型中，γ H2 AX在MMC后立即复制的DNA上过度表达，而在FANCD 2缺失的细胞中则较少。FANCD 2-耗尽的细胞也产生更少的长达240 Kb的不间断复制轨迹，无论MMC治疗如何。总的来说，数据表明，交联可能不会对S期整体造成阻碍，而是通过降低复制叉的密度并导致至少一部分叉在DNA损伤反应改变的染色质内操作来深刻地改变其进程。
DNA crosslinks can block replication in vitro and slow down S phase progression in vivo. We characterized the effect of mitomycin C crosslinker on S phase globally and on individual replication forks in wild type and FANCD2-deficient human cells. FANCD2 is critical to crosslink repair, and is also implicated in facilitating DNA replication. We used DNA fiber analysis to demonstrate persistent reduction in abundance but not progression rate of replication forks during an S phase of MMC-treated cells. FANCD2 deficiency did not eliminate this phenotype. Immunoprecipitation of EdU-labeled DNA indicated that replication was not suppressed in the domains that were undergoing response to MMC as marked by the presence of γH2AX, and in fact γH2AX was overrepresented on DNA that had replicated immediately after MMC in wild type through less so in FANCD2-depleted cells. FANCD2-depleted cells also produced fewer tracks of uninterrupted replication of up to 240Kb long, regardless of MMC treatment. Overall, the data suggest that crosslinks may not pose a block to S phase as a whole, but instead profoundly change its progress by reducing density of replication forks and causing at least a fraction of forks to operate within a DNA damage response-altered chromatin.