Allers T, Leach D R
Institute of Cell and Molecular Biology, University of Edinburgh, UK.
J Mol Biol. 1995 Sep 8;252(1):70-85. doi: 10.1006/jmbi.1994.0476.
Long DNA palindromes present a threat to genomic stability and are not tolerated in Escherichia coli. It has been suggested that this is a consequence of cruciform or hairpin formation by palindromic sequences. This work describes a methylation inhibition assay for unusual DNA secondary structure in vivo that is both internally controlled and non-invasive. If a palindrome with a central GATC target site for Dam methylase assumes a cruciform or hairpin conformation in vivo, then the GATC sequence will be located in a single-stranded loop and will consequently not be modified. The centre of a long perfect palindrome located in bacteriophage lambda is shown to be methylation-resistant in vivo. Changes to the central sequence and insertions of 10 base-pairs of asymmetric sequence do not alter the degree of under-methylation, but insertions of 20 base-pairs or more of asymmetric sequence reduce the under-methylation of the palindrome centre. We also show that the centres of long palindromes are more under-methylated than equivalent sequences in a non-palindromic context. These results are consistent with an unusual secondary structure, such as DNA cruciform or hairpin, and indicate that the formation pathway of the structure detected is independent of the composition and symmetry of the central 10 base-pairs of the palindrome.
长DNA回文序列对基因组稳定性构成威胁，在大肠杆菌中无法容忍。有人认为这是回文序列形成十字形或发夹结构的结果。这项工作描述了一种用于体内异常DNA二级结构的甲基化抑制测定法，该方法具有内部对照且非侵入性。如果一个带有Dam甲基化酶中心GATC靶位点的回文序列在体内呈现十字形或发夹构象，那么GATC序列将位于单链环中，因此不会被修饰。位于噬菌体λ中的长完美回文序列的中心在体内显示出抗甲基化。中心序列的改变和10个碱基对不对称序列的插入不会改变甲基化不足的程度，但20个碱基对或更多不对称序列的插入会降低回文序列中心的甲基化不足。我们还表明，长回文序列的中心比非回文背景下的等效序列甲基化程度更低。这些结果与异常二级结构如DNA十字形或发夹结构一致，并表明检测到的结构形成途径与回文序列中心10个碱基对的组成和对称性无关。
