Regulatory Mechanisms That Prevent Re-initiation of DNA Replication Can Be Locally Modulated at Origins by Nearby Sequence Elements

Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ∼60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome may be more susceptible to these alterations than others.真核细胞必须抑制其基因组中成千上万个复制起点处的DNA复制再起始，因为异常的复制再起始会以极高频率引发基因组变异。为了降低如此众多复制起点发生再起始的概率，细胞采用了一系列调控机制以降低复制起始蛋白的活性。鉴于这些机制的全局性，此前人们推测所有复制起点都会受到同等程度的抑制。然而，当这些机制失活时，不同复制起点的再起始效率存在显著差异，而这种差异无法通过正常S期复制过程中复制起始的效率或时序差异来解释。这一观测结果提示，可能存在另一层复制调控机制，能够对不同复制起点的再起始进行差异化调控。我们鉴定出了新型遗传元件：当复制再起始的调控被部分解除时，这些元件既是两个复制起点优先发生再起始所必需的，也足以赋予异源复制起点优先再起始的能力。该类元件并不会增强相邻复制起点在S期的复制时序或起始效率，因此它们是特异性发挥再起始启动子（re-initiation promoters，RIPs）功能的元件。我们将这两个RIP定位至约60 bp的AT富集序列，其功能发挥依赖于作用距离与序列本身。在诱导再复制的过程中，微型染色体维持复合体2-7（Mcm2-7）既会与优先发生再起始的复制起点结合，也会与不发生优先再起始的复制起点结合，这表明RIP元件能够克服Mcm2-7与复制起点结合后所施加的再起始阻断。我们的研究发现了复制再起始阻断过程中的局部调控层级。这种局部调控构建了复杂的再复制潜能基因组图谱，该图谱会在全局防再复制机制受损时显现出来。因此，正如此前针对癌细胞所推测的那样，如果再复制确实会促成基因组变异，那么基因组中的某些区域或许比其他区域更易受到这类变异的影响。