Dorn Elizabeth S, Chastain Paul D, Hall Jonathan R, Cook Jeanette Gowen
Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260, USA.
Nucleic Acids Res. 2009 Jan;37(1):60-9. doi: 10.1093/nar/gkn912. Epub 2008 Nov 14.
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.
人类细胞分裂周期面临的一项重大挑战是确保DNA复制起点不会多次启动，这种现象被称为再复制。复制控制的急性失调最终会导致广泛的DNA损伤、细胞周期检查点激活和细胞死亡，而适度失调则会促进基因组不稳定和肿瘤发生。然而，在细胞DNA含量没有可检测到的增加的情况下，很难直接证明再复制，也难以确定再复制的能力是否仅限于特定的细胞周期阶段。通过对DNA纤维铺展技术的改进，我们报告了从人类染色体的单个DNA分子上直接检测到再复制。使用这种方法，我们证明了在过量表达复制因子Cdt1的细胞进入S期1小时内存在大量再复制。此外，将HeLa癌细胞系与未转化的成纤维细胞进行比较表明，HeLa细胞在其他未受干扰的细胞周期中产生的复制信号与低水平再复制一致。在未转化的成纤维细胞系中，Cdt1抑制剂geminin耗尽后的再复制用标准检测方法无法检测到，但通过DNA纤维铺展分析很容易进行定量。对再复制的DNA分子进行直接评估将有助于增进对促进或扰乱基因组稳定性的事件的理解。
