Kim S M, Huberman J A
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, 14263, USA.
J Mol Biol. 1999 May 21;288(5):867-82. doi: 10.1006/jmbi.1999.2728.
DNA replication origins in animal cells sometimes occur in clusters. Often one of the multiple origins within these clusters fires more frequently than the others. The reason for this hierarchy remains unknown. Similar origin clusters occur in the fission yeast, Schizosaccharomyces pombe. One such cluster is located near the ura4 gene on chromosome III and contains three origins: ars3002, ars3003, and ars3004. In their natural chromosomal context (ars3003 is about 2.5 kb upstream of ars3002 and ars3004 is adjacent to ars3002 on the downstream side) their initiation frequencies display a striking hierarchy: ars3002 >> ars3003 >> ars3004. Here, we describe experiments that reveal a 400 bp replication enhancer within ars3004, adjacent to ars3002. The enhancer is essential for ars3004 origin function in a plasmid, but even with the enhancer ars3004 is an inefficient origin. The enhancer is not essential for ars3002 plasmid origin activity, but dramatically stimulates this activity, converting ars3002 from an inefficient plasmid origin to a very efficient one. It also stimulates the plasmid origin activity of ars3001 and ars3003 at all tested positions and orientations on both sides of each autonomously replicating sequence (ARS) element. If ars3002 is redefined to include the enhancer, then the relative activities of the three ARS elements as single origins within separate plasmids or as origins when all three ARS elements are present in a single plasmid is the same as the chromosomal hierarchy. Thus, this replication enhancer defines the relative activities of the three origins in the ura4 origin region. Similar enhancers may affect relative activities in the origin clusters of animal cells.
动物细胞中的DNA复制起点有时成簇出现。这些簇内的多个起点中，通常有一个比其他起点更频繁地启动复制。这种层级关系的原因尚不清楚。类似的起点簇也出现在裂殖酵母粟酒裂殖酵母中。其中一个这样的簇位于III号染色体上ura4基因附近，包含三个起点：ars3002、ars3003和ars3004。在它们的天然染色体环境中（ars3003在ars3002上游约2.5 kb处，ars3004在ars3002的下游与之相邻），它们的起始频率呈现出显著的层级关系：ars3002 >> ars3003 >> ars3004。在这里，我们描述了一些实验，这些实验揭示了ars3004内与ars3002相邻的一个400 bp的复制增强子。该增强子对于ars3004在质粒中的起点功能至关重要，但即使有了该增强子，ars3004仍然是一个低效的起点。该增强子对于ars3002质粒起点活性并非必不可少，但能显著刺激这种活性，将ars3002从一个低效的质粒起点转变为一个非常高效的起点。它还能在每个自主复制序列（ARS）元件两侧的所有测试位置和方向上刺激ars3001和ars3003的质粒起点活性。如果将ars3002重新定义为包括该增强子，那么这三个ARS元件作为单独质粒中的单个起点或当所有三个ARS元件存在于单个质粒中时作为起点的相对活性与染色体层级关系相同。因此，这种复制增强子定义了ura4起点区域中三个起点的相对活性。类似的增强子可能会影响动物细胞起点簇中的相对活性。
