Department of Molecular Biology and Genetics, University of Aarhus, Aarhus 8000, Denmark.
Nucleic Acids Res. 2013 Mar 1;41(5):3173-89. doi: 10.1093/nar/gkt051. Epub 2013 Feb 1.
To address how eukaryotic replication forks respond to fork stalling caused by strong non-covalent protein-DNA barriers, we engineered the controllable Fob-block system in Saccharomyces cerevisiae. This system allows us to strongly induce and control replication fork barriers (RFB) at their natural location within the rDNA. We discover a pivotal role for the MRX (Mre11, Rad50, Xrs2) complex for fork integrity at RFBs, which differs from its acknowledged function in double-strand break processing. Consequently, in the absence of the MRX complex, single-stranded DNA (ssDNA) accumulates at the rDNA. Based on this, we propose a model where the MRX complex specifically protects stalled forks at protein-DNA barriers, and its absence leads to processing resulting in ssDNA. To our surprise, this ssDNA does not trigger a checkpoint response. Intriguingly, however, placing RFBs ectopically on chromosome VI provokes a strong Rad53 checkpoint activation in the absence of Mre11. We demonstrate that proper checkpoint signalling within the rDNA is restored on deletion of SIR2. This suggests the surprising and novel concept that chromatin is an important player in checkpoint signalling.
为了解真核复制叉如何应对由强非共价蛋白-DNA 障碍引起的叉停滞，我们在酿酒酵母中设计了可控的 Fob-block 系统。该系统允许我们在 rDNA 内的天然位置强烈诱导和控制复制叉障碍 (RFB)。我们发现 MRX（Mre11、Rad50、Xrs2）复合物在 RFB 处对叉完整性起着关键作用，这与它在双链断裂处理中的公认功能不同。因此，在没有 MRX 复合物的情况下，rDNA 处会积累单链 DNA (ssDNA)。基于此，我们提出了一个模型，即 MRX 复合物专门保护蛋白-DNA 障碍处停滞的叉，而其缺失会导致加工产生 ssDNA。令我们惊讶的是，这种 ssDNA 不会引发检查点反应。然而，令人好奇的是，在没有 Mre11 的情况下，将 RFB 异位放置在染色体 VI 上会引发强烈的 Rad53 检查点激活。我们证明，在删除 SIR2 后，rDNA 内的适当检查点信号得到恢复。这表明了一个令人惊讶和新颖的概念，即染色质是检查点信号的重要参与者。
