Mukhopadhyay G, Chattoraj D K
Laboratory of Biochemistry, NCI, NIH, Bethesda, MD 20892.
J Mol Biol. 1993 May 5;231(1):19-28. doi: 10.1006/jmbi.1993.1253.
The origin of plasmid DNA replication in bacteriophage P1 has five 19 base-pair sites that bind the plasmid-encoded initiator, RepA. Here we show, using a DNA band retardation assay, that RepA can bend DNA that carries one or more of the RepA binding sites. RepA binding to supercoiled DNA carrying the five sites, directly repeated and phased two turns of B-DNA apart, absorbs about one positive superhelical turn of DNA as determined by two-dimensional gel electrophoresis. This indicates that the DNA is wrapped around RepA as a consequence of in-phase bending at the individual binding sites. The RepA-DNA complexes did not show elevated sensitivity to KMnO4, a reagent specific for pyrimidine bases (T >> C) in unstacked DNA. The wrapping of the DNA around RepA, therefore, does not lead to significant unwinding of the double helix. Extensive unwinding, suitable for the initiation of DNA replication, most likely requires participation of factors other than RepA. We also noted that the thymine bases of the sequence 5'-ATC-3', of which there are 20 in the origin, all reacted to KMnO4 strongly whether or not RepA was present. The preferential reactivity of ATC sequences was specific to the origin region, as thymine residues including those in the ATC sequences did not display elevated sensitivity to KMnO4 in a DNA fragment from pBR322. On one of two strands of the origin the selective reactivity at the ATC sequences was supercoiling dependent. These results indicate that the origin includes unstacked DNA bases, the significance of which remains to be determined.
噬菌体P1中质粒DNA复制的起始位点有五个19个碱基对的位点，可结合质粒编码的起始蛋白RepA。在此我们利用DNA条带阻滞分析表明，RepA能够使携带一个或多个RepA结合位点的DNA发生弯曲。RepA与携带这五个位点的超螺旋DNA结合，这些位点直接重复且相隔两圈B型DNA排列，通过二维凝胶电泳测定，RepA结合会吸收大约一圈正超螺旋的DNA。这表明由于在各个结合位点的同相弯曲，DNA缠绕在RepA周围。RepA-DNA复合物对KMnO₄（一种对未堆积DNA中的嘧啶碱基（T >> C）具有特异性的试剂）没有表现出更高的敏感性。因此，DNA围绕RepA的缠绕不会导致双螺旋的显著解旋。广泛的解旋，适合于DNA复制的起始，很可能需要RepA以外的其他因子参与。我们还注意到，起始位点序列5'-ATC-3'中的胸腺嘧啶碱基，起始位点中有20个这样的碱基，无论RepA是否存在，它们都与KMnO₄发生强烈反应。ATC序列的优先反应性是起始区域特有的，因为包括ATC序列中的胸腺嘧啶残基在内，pBR322 DNA片段中的胸腺嘧啶残基对KMnO₄没有表现出更高的敏感性。在起始位点两条链中的一条上，ATC序列的选择性反应性依赖于超螺旋。这些结果表明起始位点包括未堆积的DNA碱基，其意义尚待确定。
