Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome [array]. Saccharomyces cerevisiae

Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4∆ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1∆ cells, and checkpoint-defective mec1∆ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres. Overall design: 30 total samples: (6 samples - BrdU- HU arrest 45min with 2 replicates, strains: WT, rif1 delta, pfa4 delta) (12 samples -S-phase BrdU time course with 2 replicates at 25 and 35 min, strains: WT, rif1 delta, mec1_100) (12 samples - S-phase BrdU time course with 2 replicates at 25 and 35 min, strains: sml1 delta, sml1 delta rif1 delta, sml1 delta mec1 delta)

染色体DNA复制需数百至数千个复制起点（replication origin）协同激活。单个复制起点在S期受到表观遗传调控，使得S期内复制起始的效率与启动时序存在差异。现有研究表明，该调控过程与染色质结构及组织形成的时序结构域相关，这类结构域招募限制性复制因子的能力各不相同。近年来，Rif1已在裂殖酵母与哺乳动物细胞中被鉴定为全基因组范围的复制时序调控因子。然而，此前芽殖酵母中的研究提示，Rif1在复制时序调控中的作用或仅局限于亚端粒结构域，且其功能源于已知的端粒长度调控机制。本研究通过全基因组分析5-溴-2'-脱氧尿苷（BrdU）掺入情况解析复制时序，结果显示，Rif1可调控酿酒酵母（S. cerevisiae）全基因组范围内晚期/休眠复制起点的复制时序。对存在Rif1棕榈酰化与膜结合缺陷的pfa4Δ细胞的分析表明，Rif1对复制时序的调控，与其将端粒锚定至核膜周边的功能相互独立。rif1Δ细胞内的S期检验点信号通路完好，且检验点缺陷的mec1Δ细胞并未出现同等程度的复制时序失调，这共同证明Rif1通过不依赖于该检验点的机制调控复制时序。本研究结果显示，Rif1的调控机制可独立于与染色体末端的距离影响复制起点的时序，反而提示端粒序列仅为招募Rif1的蛋白提供了丰富的结合位点。不过，端粒结构域内丰富的Rif1结合位点，或许有助于Rif1介导的、对着丝粒远端非端粒复制起点的抑制作用。整体实验设计：共包含30份样本：1. 6份样本：采用BrdU标记联合羟基脲（HU）阻滞45分钟，设置2次生物学重复，受试菌株为野生型（WT）、rif1Δ、pfa4Δ；2. 12份样本：S期BrdU时间进程实验，分别在25分钟与35分钟两个时间点设置2次生物学重复，受试菌株为WT、rif1Δ、mec1_100；3. 12份样本：S期BrdU时间进程实验，分别在25分钟与35分钟两个时间点设置2次生物学重复，受试菌株为sml1Δ、sml1Δ rif1Δ、sml1Δ mec1Δ。