Richardson Christopher D, Li Joachim J
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America.
Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, United States of America.
PLoS Genet. 2014 Jun 19;10(6):e1004358. doi: 10.1371/journal.pgen.1004358. eCollection 2014 Jun.
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数千个起始位点处的DNA复制重新起始，因为重新起始会以极高的频率产生基因组改变。为了将这么多起始位点重新起始的可能性降至最低，细胞使用了一系列调节机制来降低复制起始蛋白的活性。鉴于这些机制的全局性，人们推测所有起始位点受到的抑制是相同的。然而，当这些机制失效时，不同起始位点重新起始的效率各异，而这种差异无法用正常S期复制期间起始位点起始的效率或时间差异来解释。这一观察结果提示存在另一层复制控制，它可以在不同起始位点上差异性地调节重新起始。我们已经鉴定出了新的遗传元件，它们对于两个起始位点的优先重新起始是必需的，并且当重新起始的控制部分解除调控时，足以赋予异源起始位点优先重新起始的能力。这些元件不会提高相邻起始位点的S期时间或效率，因此它们专门作为重新起始启动子（RIPs）发挥作用。我们已经将这两个RIPs定位到约60 bp富含AT的序列上，这些序列以距离和序列依赖性的方式起作用。在诱导重新复制的过程中，Mcm2-7既与优先重新起始的起始位点重新结合，也与不优先重新起始的起始位点重新结合，这表明RIP元件可以克服Mcm2-7与起始位点结合后施加的重新起始障碍。我们的发现确定了重新起始障碍中的局部控制水平。当防止重新复制的全局机制受损时，这种局部控制会产生一个复杂的重新复制潜力基因组格局。因此，如果重新复制确实如推测的那样导致癌细胞的基因组改变，那么基因组的某些区域可能比其他区域更容易受到这些改变的影响。
