在不稳定反向重复序列处的复制停滞：DNA发夹结构与叉状稳定蛋白之间的相互作用
Replication stalling at unstable inverted repeats: interplay between DNA hairpins and fork stabilizing proteins.
作者信息
Voineagu Irina, Narayanan Vidhya, Lobachev Kirill S, Mirkin Sergei M
机构信息
Department of Biology, Tufts University, Medford, MA 02155, USA.
出版信息
Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):9936-41. doi: 10.1073/pnas.0804510105. Epub 2008 Jul 15.
DNA inverted repeats (IRs) are hotspots of genomic instability in both prokaryotes and eukaryotes. This feature is commonly attributed to their ability to fold into hairpin- or cruciform-like DNA structures interfering with DNA replication and other genetic processes. However, direct evidence that IRs are replication stall sites in vivo is currently lacking. Here, we show by 2D electrophoretic analysis of replication intermediates that replication forks stall at IRs in bacteria, yeast, and mammalian cells. We found that DNA hairpins, rather than DNA cruciforms, are responsible for the replication stalling by comparing the effects of specifically designed imperfect IRs with varying lengths of their central spacer. Finally, we report that yeast fork-stabilizing proteins, Tof1 and Mrc1, are required to counteract repeat-mediated replication stalling. We show that the function of the Tof1 protein at DNA structure-mediated stall sites is different from its previously described effect on protein-mediated replication fork barriers.
DNA反向重复序列（IRs）是原核生物和真核生物基因组不稳定的热点区域。这一特征通常归因于它们折叠成发夹状或十字形DNA结构的能力，这种结构会干扰DNA复制和其他遗传过程。然而，目前缺乏IRs在体内是复制停滞位点的直接证据。在这里，我们通过对复制中间体的二维电泳分析表明，在细菌、酵母和哺乳动物细胞中，复制叉在IRs处停滞。通过比较特定设计的具有不同长度中心间隔区的不完全IRs的影响，我们发现DNA发夹而非DNA十字形结构是导致复制停滞的原因。最后，我们报告酵母叉稳定蛋白Tof1和Mrc1是抵消重复序列介导的复制停滞所必需的。我们表明，Tof1蛋白在DNA结构介导的停滞位点的功能与其先前描述的对蛋白质介导的复制叉屏障的作用不同。
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