Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase.
Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase.
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DOI：
10.1101/gr.196857.115
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发表时间：
2016-03
期刊：
影响因子：
7
通讯作者：
Aparicio OM
中科院分区：
文献类型：
--
作者：
Peace JM;Villwock SK;Zeytounian JL;Gan Y;Aparicio OM
酿酒酵母 Forkhead Box (FOX) 蛋白 Fkh1 和 Fkh2 调节多种细胞过程，包括转录、同源重组过程中的长程 DNA 相互作用、复制起点计时和长程起点聚类。我们假设，作为早期起源激活的刺激物，Fkh1 和 Fkh2 丰度限制了全基因组起源激活的速率。然而，现有方法不太适合对菌株和条件之间的起源激发进行定量、全基因组测量。为了克服这一限制，我们开发了 qBrdU-seq，一种用于复制动态的 BrdU 掺入分析的定量方法，并应用它来表明 Fkh1 和 Fkh2 的过度表达提前了整个基因组中许多起点的起始时间，从而导致早期 S 期起点起始的总水平更高。较高的起始速率伴随着较慢的复制叉进展，从而维持正常的 S 期长度，而不会引起可检测到的 Rad53 检查点激酶激活。原点发射时间的提前，包括异色域中原点的提前，是在 G1 阶段后期建立的，这表明原点计时可以在原点许可之后重置。这些结果通过将 Fkh1 和 Fkh2 确定为起点激发的速率限制因素，为起点计时调控机制提供了新的见解，这些因素决定了复制起点产生限制因素的能力，并有可能在 G1 期后期重新编程复制计时。
The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of early origin activation, Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, are not well-suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advances the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase.