Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin, which extends up to 1 kb from early, efficient replication origins. The CMG holohelicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

中文翻译：

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在 DNA 复制缺失的情况下，通过解旋酶激活来破坏原始染色质结构

在 DNA 复制开始之前，必须激活真核解旋酶 Mcm2-7，以在早期 S 期的复制起始位点解旋 DNA。为了研究原始染色质内解旋酶的激活，我们构建了聚合酶 α 亚基 Cdc17（或 Pol1）的条件突变体，以防止启动并阻止复制。这些细胞在允许的条件下恢复导致在起点处产生未复制的间隙，这可能是由于复制开始之前解旋酶的激活所致。我们在限制条件下使用基于微球菌核酸酶 (MNase) 的染色质占用分析来研究与解旋酶激活相关的染色质动力学。在 DNA 复制缺失的情况下解旋酶激活会导致染色质的破坏和解体，染色质从早期有效的复制起点延伸至 1 kb。CMG 全解旋酶复合物也从起点移出相同的距离，产生激活 S 相内检查点的单链 DNA。检查点的丢失并不能调节 CMG 复合物的进展和停滞，而是导致早期和晚期起源的染色质破坏。最后，我们发现，在没有 DNA 复制的情况下，局部序列背景调节解旋酶的进展，这表明解旋酶在与复制脱钩时本质上的进行性较低。