Görisch Sabine M, Sporbert Anje, Stear Jeffrey H, Grunewald Ingrid, Nowak Danny, Warbrick Emma, Leonhardt Heinrich, Cardoso M Cristina
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Cell Cycle. 2008 Jul 1;7(13):1983-90. doi: 10.4161/cc.7.13.6094. Epub 2008 Apr 11.
The precise coordination of the different steps of DNA replication is critical for the maintenance of genome stability. We have probed the mechanisms coupling various components of the replication machinery and their response to polymerase stalling by inhibition of the DNA polymerases in living mammalian cells with aphidicolin. We observed little change in the behaviour of proteins involved in the initiation of DNA replication. In contrast, we detected a marked accumulation of the single stranded DNA binding factor RPA34 at sites of DNA replication. Finally, we demonstrate that proteins involved in the elongation step of DNA synthesis dissociate from replication foci in the presence of aphidicolin. Taken together, these data indicate that inhibition of processive DNA polymerases uncouples the initiation of DNA replication from subsequent elongation steps. We, therefore, propose that the replication machinery is made up of distinct functional sub-modules that allow a flexible and dynamic response to challenges during DNA replication.
DNA复制不同步骤的精确协调对于维持基因组稳定性至关重要。我们通过用阿非迪霉素抑制活的哺乳动物细胞中的DNA聚合酶，探究了复制机制中各种组分的偶联机制及其对聚合酶停滞的反应。我们观察到参与DNA复制起始的蛋白质行为几乎没有变化。相比之下，我们在DNA复制位点检测到单链DNA结合因子RPA34显著积累。最后，我们证明在阿非迪霉素存在下，参与DNA合成延伸步骤的蛋白质从复制灶解离。综上所述，这些数据表明持续性DNA聚合酶的抑制使DNA复制的起始与后续延伸步骤解偶联。因此，我们提出复制机制由不同的功能子模块组成，这些子模块允许对DNA复制过程中的挑战做出灵活而动态的反应。
