Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast. Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast

Ribosomal DNA (rDNA) is organized as large arrays of tandem repeats that vary in copy number from a few dozen to hundreds. In the budding yeast Saccharomyces cerevisiae, each rDNA repeat includes a potential origin of replication. Previous work has led to the model that the rDNA replication origins compete for limiting replication initiation factors with origins in the rest of the genome, suggesting that reduction in rDNA copy number would reduce competition for these limiting factors and therefore promote origin usage in the rest of the genome. To test this hypothesis, we compared genome-wide replication in strains with either wild type rDNA copy number of ~180 (“180 rDNA”) or just ~35 copies (“35 rDNA”) by performing dense-to-light isotope transfer experiments to physically separate replicated, hybrid-density (HL or heavy-light) DNA from unreplicated, HH (heavy-heavy) DNA in cell samples collected at different times in S phase. Contrary to our expectations, we find that although there are no apparent differences in non-rDNA origin activity between the two strains, the 35 rDNA strain shows a genome-wide delay in progression through S phase compared to the 180 rDNA strain. Overall design: To profile genome-wide replication, we grew cells in isotopically dense (13C-glucose, 15N-ammonium sulfate) for ≥7 population doublings. We then arrested the cells in G1 using a-factor, filtered and resuspended the cells in isotopically light (12C, 14N) medium, then released them into S phase by addition of Pronase. We collected samples at intervals through the ensuing S phase (30,35,40,45,50 min for 35 rDNA strain; 25,30,35,40,45,55 min for 180 rDNA strain). We extracted the DNA, and digested it with a restriction enzyme. Unreplicated (HH) DNA was separated from replicated (HL) DNA by ultracentrifugation in cesium chloride gradients, which were subsequently drip-fractionated. Small aliquots of each fraction were hybridized on a slot blot with a [32P]-labeled whole-genome probe, both to identify fractions with HH and HL DNA and to quantify the percent replication in each S phase sample. For each sample, we then pooled all the HH fractions and labeled the DNA with Cy3, likewise labeled the HL DNA with Cy5, and co-hybridized the pair to a microarray. After extraction of hybridization intensities, we used the known % replication values from the slot blot analysis to normalize the overall % replication in each sample, with an added correction for the percentage of cells in the population that successfully completed S phase (obtained from the maximum % replication seen at the end of S phase from the slot blot analysis). The normalized % replication values were loess-smoothed with a window of 22 kb and plotted as a function of chromosome coordinate with a step size of 500 bp. For a detailed description of this protocol, see Peng et al. (2014), Methods Mol Biol 1170:477-99 (PMCID: PMC4338859).

核糖体DNA（ribosomal DNA，rDNA）以串联重复的大型阵列形式存在，拷贝数范围从数十到数百不等。在出芽酵母酿酒酵母（Saccharomyces cerevisiae）中，每个rDNA重复序列均包含一个潜在的复制起点。既往研究提出了如下模型：rDNA复制起点会与基因组其他区域的复制起点竞争有限的复制起始因子，据此推测rDNA拷贝数减少可降低对这类有限因子的竞争，进而促进基因组其他区域的复制起点激活。为验证该假说，我们采用重-轻同位素转移实验，对S期不同时间点收集的细胞样本进行处理，将复制产生的杂交密度（HL，即重-轻）DNA与未复制的HH（重-重）DNA物理分离，以此比较rDNA拷贝数分别为野生型水平（约180个，记为"180 rDNA"）和仅约35个（记为"35 rDNA"）的两株菌株的全基因组复制情况。

与我们的预期相悖，尽管两株菌株的非rDNA复制起点活性无显著差异，但与"180 rDNA"菌株相比，"35 rDNA"菌株的全基因组S期进程出现了明显延迟。

总体实验设计：为解析全基因组复制特征，我们将细胞在同位素重培养基（13C-葡萄糖、15N-硫酸铵）中培养至少7个世代。随后利用α因子将细胞阻滞于G1期，过滤后重悬于同位素轻培养基（12C、14N）中，通过加入链霉蛋白酶（Pronase）使细胞同步进入S期。在后续S期进程中按间隔时间收集样本："35 rDNA"菌株的收集时间点为30、35、40、45、50 min；"180 rDNA"菌株为25、30、35、40、45、55 min。提取细胞DNA后用限制性内切酶进行酶切。通过氯化铯梯度超速离心，将未复制的HH DNA与复制的HL DNA分离，随后对梯度进行滴式分部收集。取各分部的少量样本，使用[32P]标记的全基因组探针进行斑点杂交，既可鉴定含有HH和HL DNA的分部，也可量化每个S期样本的复制百分比。对于每个样本，我们合并所有HH分部的DNA，用Cy3进行荧光标记；同理将HL DNA用Cy5标记，随后将两种标记的DNA共杂交至微阵列芯片。获取杂交强度数据后，我们利用斑点杂交分析得到的已知复制百分比值，对每个样本的总复制百分比进行标准化，并针对群体中成功完成S期的细胞占比进行额外校正（该占比由斑点杂交分析中S期结束时的最大复制百分比获得）。将标准化后的复制百分比值以22 kb为窗口进行局部加权回归（loess）平滑，并以500 bp为步长，按染色体坐标绘制函数曲线。关于该实验流程的详细描述，参见Peng等人（2014），《Methods Mol Biol》1170:477-99（PMCID: PMC4338859）。