Tanaka Taku, Nishito Yasumasa, Masai Hisao
Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, 4-6-1 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Basic Technology Research Center, Tokyo Metropolitan Institute of Medical Science, 4-6-1 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Biochem Biophys Res Commun. 2016 Feb 12;470(3):546-551. doi: 10.1016/j.bbrc.2016.01.108. Epub 2016 Jan 20.
Arrest of replication fork progression is one of the most common causes for increasing the genomic instability. In bacteria, PriA, a conserved DEXH-type helicase, plays a major role in recognition of the stalled forks and restart of DNA replication. We took advantage of PriA's ability to specifically recognize stalled replication forks to determine the genomic loci where replication forks are prone to stall on the Escherichia coli genome. We found that PriA binds around oriC upon thymine starvation which reduces the nucleotide supply and causes replication fork stalling. PriA binding quickly disappeared upon readdition of thymine. Furthermore, BrdU was incorporated at around oriC upon release from thymine starvation. Our results indicate that reduced supply of DNA replication precursors causes replication fork stalling preferentially in the 600 kb segment centered at oriC. This suggests that replication of the vicinity of oriC requires higher level of nucleotide precursors. The results also point to a possibility of slow fork movement and/or the presence of multiple fork arrest signals within this segment. Indeed, we have identified rather strong fork stall/pausing signals symmetrically located at ∼50 kb away from oriC. We speculate that replication pausing and fork-slow-down shortly after initiation may represent a novel checkpoint that ensures the presence of sufficient nucleotide supply prior to commitment to duplication of the entire genome.
复制叉进展停滞是基因组不稳定性增加的最常见原因之一。在细菌中，PriA是一种保守的DEXH型解旋酶，在识别停滞的复制叉和重启DNA复制中起主要作用。我们利用PriA特异性识别停滞复制叉的能力来确定大肠杆菌基因组上复制叉易于停滞的基因组位点。我们发现，在胸腺嘧啶饥饿时，PriA结合在oriC周围，这会减少核苷酸供应并导致复制叉停滞。重新添加胸腺嘧啶后，PriA的结合迅速消失。此外，从胸腺嘧啶饥饿状态释放后，BrdU掺入oriC周围。我们的结果表明，DNA复制前体供应减少会导致复制叉优先在以oriC为中心的600 kb片段中停滞。这表明oriC附近的复制需要更高水平的核苷酸前体。结果还指出了该片段内复制叉移动缓慢和/或存在多个复制叉停滞信号的可能性。事实上，我们已经确定了在距oriC约50 kb处对称定位的相当强的复制叉停滞/暂停信号。我们推测，起始后不久的复制暂停和复制叉减速可能代表一种新的检查点，可确保在承诺复制整个基因组之前有足够的核苷酸供应。
