Analysis of re-replication from deregulated origin licensing by DNA fiber spreading.
Analysis of re-replication from deregulated origin licensing by DNA fiber spreading.
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DOI：
10.1093/nar/gkn912
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发表时间：
2009-01
影响因子：
14.9
通讯作者：
Cook, Jeanette Gowen
中科院分区：
文献类型：
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作者：
Dorn, Elizabeth S.;Chastain, Paul D., II;Hall, Jonathan R.;Cook, Jeanette Gowen
每个人类细胞分裂周期的一个主要挑战是确保DNA复制起点不会启动一次以上，这种现象称为再复制。复制控制的急性失调最终导致广泛的DNA损伤，细胞周期检查点激活和细胞死亡，而中度失调促进基因组不稳定和肿瘤发生。然而，在细胞DNA含量没有可检测的增加的情况下，难以直接证明再复制或确定再复制的能力是否限于特定的细胞周期阶段。使用适应的DNA纤维的传播，我们报告的直接检测从人类染色体上的单个DNA分子的再复制。使用这种方法，我们证明大量的再复制1小时内的S期进入细胞过度生产的复制因子，Cdt1。此外，HeLa癌细胞系与未转化的成纤维细胞的比较表明，HeLa细胞产生的复制信号与在其他未受干扰的细胞周期中的低水平再复制一致。在未转化的成纤维细胞系中，Cdt1抑制剂geminin耗尽后的再复制通过标准测定法检测不到，但通过DNA纤维铺展分析可容易地定量。直接评估重新复制的DNA分子将促进对促进或干扰基因组稳定性的事件的理解。
A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability.
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影响因子：
5.3
作者：
Ishida, S;Huang, E;Nevins, JR
通讯作者：
Nevins, JR
影响因子：
5.3
作者：
Braden, Wesley A.;Lenihan, Jon M.;Knudsen, Erik S.
通讯作者：
Knudsen, Erik S.
影响因子：
3.7
作者：
Conti, Chiara;Herrick, John;Bensimon, Aaron
通讯作者：
Bensimon, Aaron
影响因子：
10.5
作者：
Gomez, Maria;Antequera, Francisco
通讯作者：
Antequera, Francisco
DOI：
10.1038/nrm1663
发表时间：
2005-06
期刊：
Nature reviews. Molecular cell biology
影响因子：
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作者：
通讯作者：
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