Zhang S, Seeman N C
Department of Chemistry, New York University, New York, NY 10003.
J Mol Biol. 1994 May 20;238(5):658-68. doi: 10.1006/jmbi.1994.1327.
The Holliday junction is a four-stranded branched DNA structure found as an intermediate in genetic recombination. Naturally occurring Holliday junctions have homologous (2-fold) sequence symmetry; this symmetry renders the site of the branch unstable, because the molecules can undergo an isomerization called branch migration. For the past decade, these molecules have been modeled in non-migrating "immobile" branched junctions lacking this symmetry. The solution structure derived from immobile branched junctions is a two-domain DNA structure, in which a particular pair of strands forms the crossover between domains, and the other pair of strands has a structure similar to that seen in DNA double helices; reversal of these roles has not been apparent. We investigate here whether this bias ("crossover preference") for particular strands to be the crossover pair extends to structures that contain symmetric sequences flanking the branch point. We employ nicked symmetric immobile junctions in a competition assay to determine the extent of crossover preference. We have measured all six of the 2-fold symmetric dinucleotide sequences that can flank a branch point, and find small preferences (< 650 cal/mol) at 4 degrees C. The largest preference decreases when the 2-fold symmetry is extended to tetranucleotides. The system contains an internal control on systematic errors, because there are 4-fold symmetric dinucleotide sequences that should show no bias; the preferences we measure for them are below or similar to our estimated errors. We have calibrated our experiment by measuring the crossover preferences for a previously characterized immobile junction; the preference is slightly larger than the largest preference seen for a symmetric crossover. We conclude that large crossover preferences are not characteristic of junctions with extensive symmetry, and are thus unlikely to have important consequences for genetic recombination.
霍利迪连接体是一种四链分支DNA结构，是遗传重组过程中的中间体。天然存在的霍利迪连接体具有同源（2倍）序列对称性；这种对称性使分支位点不稳定，因为分子可以发生一种称为分支迁移的异构化。在过去十年中，这些分子已被模拟为缺乏这种对称性的非迁移性“固定”分支连接体。从固定分支连接体推导出来的溶液结构是一种双结构域DNA结构，其中特定的一对链在结构域之间形成交叉，另一对链具有与DNA双螺旋中所见结构相似的结构；这些作用的逆转并不明显。我们在此研究这种特定链作为交叉对的偏向性（“交叉偏好”）是否延伸到在分支点两侧包含对称序列的结构。我们在竞争试验中使用带切口的对称固定连接体来确定交叉偏好的程度。我们测量了可以位于分支点两侧的所有六种2倍对称二核苷酸序列，发现在4摄氏度时有小的偏好（<650卡/摩尔）。当2倍对称性扩展到四核苷酸时，最大偏好性降低。该系统包含对系统误差的内部控制，因为存在应无偏向性的4倍对称二核苷酸序列；我们测量到的它们的偏好低于或类似于我们估计的误差。我们通过测量先前表征的固定连接体的交叉偏好来校准我们的实验；该偏好略大于对称交叉所见的最大偏好。我们得出结论，大的交叉偏好不是具有广泛对称性的连接体的特征，因此不太可能对遗传重组产生重要影响。
