Pal S K, Mason R J, Chattoraj D K
J Mol Biol. 1986 Nov 20;192(2):275-85. doi: 10.1016/0022-2836(86)90364-5.
The copy number control locus incA of unit copy plasmid P1 maps in a region containing nine 19 base-pair repeats. Previous results from studies in vivo and in vitro indicated that incA interacts with the plasmid-encoded RepA protein, which is essential for replication. It has been proposed that the repeat sequences negatively control copy number by sequestering the RepA protein, which is rate-limiting for replication. Our results lend further support to this hypothesis. Here we show that the repeats can be deleted completely from P1 miniplasmids and the deletion results in an approximately eightfold increase in plasmid copy number. So, incA sequences are totally dispensable for replication and have only a regulatory role. The copy number of incA-deleted plasmids can be reduced if incA sequences are present in trans or are reincorporated at two different positions in the plasmid. This reduction in copy number is not due to lowered expression of the repA gene in the presence of incA. We show that one repeat sequence is sufficient to bind RepA and can reduce the copy number of incA-deleted plasmids. When part of the repeat was deleted, it lost its ability to bind as well as influence copy number. These results show a strong correlation between the capacity of incA repeats to bind RepA protein both in vivo and in vitro, and the function of incA in the control of copy number.
单拷贝质粒P1的拷贝数控制基因座incA定位于一个包含9个19碱基对重复序列的区域。先前体内和体外研究结果表明，incA与质粒编码的RepA蛋白相互作用，而RepA蛋白对复制至关重要。有人提出，重复序列通过隔离对复制起限速作用的RepA蛋白来负向控制拷贝数。我们的结果进一步支持了这一假说。在此我们表明，重复序列可从P1微型质粒中完全删除，且删除后质粒拷贝数增加约8倍。因此，incA序列对于复制完全是可有可无的，仅具有调控作用。如果incA序列以反式存在或重新整合到质粒中的两个不同位置，则incA缺失质粒的拷贝数会降低。拷贝数的这种降低并非由于在存在incA的情况下repA基因表达降低所致。我们表明，一个重复序列就足以结合RepA，并能降低incA缺失质粒的拷贝数。当部分重复序列被删除时，它就失去了结合以及影响拷贝数的能力。这些结果表明，incA重复序列在体内和体外结合RepA蛋白的能力与incA在拷贝数控制中的功能之间存在很强的相关性。
