Gray Steven J, Liu Guoqi, Altman Amy L, Small Lawrence E, Fanning Ellen
Department of Biological Sciences, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37235-1634, USA.
Exp Cell Res. 2007 Jan 1;313(1):109-20. doi: 10.1016/j.yexcr.2006.09.020. Epub 2006 Sep 28.
The Chinese hamster dihydrofolate reductase (DHFR) DNA replication initiation region, the 5.8 kb ori-beta, can function as a DNA replicator at random ectopic chromosomal sites in hamster cells. We report a detailed genetic analysis of the DiNucleotide Repeat (DNR) element, one of several sequence elements necessary for ectopic ori-beta activity. Deletions within ori-beta identified a 132 bp core region within the DNR element, consisting mainly of dinucleotide repeats, and a downstream region that are required for ori-beta initiation activity at non-specific ectopic sites in hamster cells. Replacement of the DNR element with Xenopus or mouse transcriptional elements from rDNA genes restored full levels of initiation activity, but replacement with a nucleosome positioning element or a viral intron sequence did not. The requirement for the DNR element and three other ori-beta sequence elements was conserved when ori-beta activity was tested at either random sites or at a single specific ectopic chromosomal site in human cells. These results confirm the importance of specific cis-acting elements in directing the initiation of DNA replication in mammalian cells, and provide new evidence that transcriptional elements can functionally substitute for one of these elements in ori-beta.
中国仓鼠二氢叶酸还原酶（DHFR）DNA复制起始区域，即5.8 kb的ori-β，可在仓鼠细胞的随机异位染色体位点发挥DNA复制起点的功能。我们报告了对双核苷酸重复（DNR）元件的详细遗传分析，该元件是异位ori-β活性所需的几个序列元件之一。ori-β内的缺失确定了DNR元件内一个132 bp的核心区域，主要由双核苷酸重复序列组成，以及一个下游区域，这两个区域是仓鼠细胞中非特异性异位位点ori-β起始活性所必需的。用来自rDNA基因的非洲爪蟾或小鼠转录元件替换DNR元件可恢复全部起始活性水平，但用核小体定位元件或病毒内含子序列替换则不能。当在人类细胞的随机位点或单个特定异位染色体位点测试ori-β活性时，对DNR元件和其他三个ori-β序列元件的需求是保守的。这些结果证实了特定顺式作用元件在指导哺乳动物细胞中DNA复制起始方面的重要性，并提供了新的证据表明转录元件可以在功能上替代ori-β中的这些元件之一。
