Kehrli Keffy R M, Sidorova Julia M
Department of Pathology, University of Washington, Seattle, WA 98195.
Oncoscience. 2014;1(7):540-555. doi: 10.18632/oncoscience.70.
DNA crosslinks can block replication and slow down S phase progression . 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.
DNA交联可阻断复制并减缓S期进程。我们在野生型和FANCD2缺陷型人类细胞中，全面表征了丝裂霉素C交联剂对S期及单个复制叉的影响。FANCD2对交联修复至关重要，并且也参与促进DNA复制。我们使用DNA纤维分析来证明，在丝裂霉素C处理的细胞的S期，复制叉的丰度持续降低，但进展速率未受影响。FANCD2缺陷并未消除这种表型。对EdU标记的DNA进行免疫沉淀表明，在以γH2AX的存在为标志的对丝裂霉素C产生反应的区域中，复制并未受到抑制，实际上在野生型细胞中，γH2AX在丝裂霉素C处理后立即复制的DNA上的比例过高，而在FANCD2缺失的细胞中比例较低。无论是否进行丝裂霉素C处理，FANCD2缺失的细胞产生的长达240Kb的不间断复制轨迹也较少。总体而言，数据表明交联可能不会对整个S期造成阻断，而是通过降低复制叉密度并导致至少一部分复制叉在DNA损伤反应改变的染色质内运行，从而深刻改变其进程。
