Guseva Serafima, Szekely Or, Geng Ainan, Smith Kai, Pratihar Supriya, Gu Stephanie, Al-Hashimi Hashim M
bioRxiv. 2024 Dec 23:2024.12.22.630000. doi: 10.1101/2024.12.22.630000.
In duplex DNA, A-T and G-C form Watson-Crick base pairs, and Hoogsteen pairing only dominates upon protein binding or DNA damage. Using NMR, we show that an A-T Hoogsteen base pair previously observed in crystal structures of transposon DNA hairpins bound to TnpA protein forms in solution even in the absence of TnpA. This Hoogsteen base pair, located adjacent to a dinucleotide apical loop, exists in dynamic equilibrium with a minor Watson-Crick conformation (population ∼11% and lifetime ∼55 µs). Extending the apical loop to three residues inverted the equilibrium, making Watson-Crick the dominant state and the Hoogsteen conformation recognized by TnpA a minor state (population ∼14% and lifetime ∼28 µs). The propensity for Hoogsteen pairing depended on apical loop residues, which form contacts directly or indirectly stabilizing the Hoogsteen conformation. A structure survey did not reveal Hoogsteen pairing near RNA apical loops making them unique to DNA. Our results demonstrate that Hoogsteen can be the dominant state even in naked unmodified duplex DNA and identify 5'-CTT(T/C)AG-3' as a DNA-specific apical loop motif stabilized by Hoogsteen pairing. Hoogsteen base pairs may be prevalent in DNA hairpins forming during replication and transcription, with broad implications for the genomic landscape.
在双链DNA中，A-T和G-C形成沃森-克里克碱基对，只有在蛋白质结合或DNA损伤时，霍格施泰因配对才占主导。我们利用核磁共振表明，先前在与TnpA蛋白结合的转座子DNA发夹晶体结构中观察到的A-T霍格施泰因碱基对，即使在没有TnpA的情况下也能在溶液中形成。这个霍格施泰因碱基对位于一个二核苷酸顶端环的旁边，与一个次要的沃森-克里克构象处于动态平衡（丰度约为11%，寿命约为55微秒）。将顶端环延伸至三个残基会使平衡反转，使沃森-克里克构象成为主要状态，而被TnpA识别的霍格施泰因构象成为次要状态（丰度约为14%，寿命约为28微秒）。霍格施泰因配对的倾向取决于顶端环残基，这些残基直接或间接形成稳定霍格施泰因构象的接触。一项结构调查未在RNA顶端环附近发现霍格施泰因配对，这使得它们成为DNA特有的结构。我们的结果表明，即使在未修饰的裸双链DNA中，霍格施泰因配对也可以是主要状态，并确定5'-CTT(T/C)AG-3'为通过霍格施泰因配对稳定的DNA特异性顶端环基序。霍格施泰因碱基对可能在复制和转录过程中形成的DNA发夹中普遍存在，对基因组格局具有广泛影响。
