Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, United States.
Biochemistry. 2012 Mar 27;51(12):2407-16. doi: 10.1021/bi201710q. Epub 2012 Mar 14.
Ribonucleotides are frequently incorporated into DNA during replication. They are recognized and processed by several cellular enzymes, and their continued presence in the yeast nuclear genome results in replicative stress and genome instability. Thus, it is important to understand the effects of isolated ribonucleotide incorporation on DNA structure. With this goal in mind, we describe the nuclear magnetic resonance structure of the self-complementary Dickerson dodecamer sequence d(CGC)rGd(AATTCGCG) containing two symmetrically positioned riboguanosines. The absence of an observable H(1)-H(2) scalar coupling interaction indicates a C3'-endo conformation for the ribose. Longer-range structural perturbations resulting from the presence of the ribonucleotide are limited to the adjacent and transhelical nucleotides, while the global B-form DNA structure is maintained. Because crystallographic studies have indicated that isolated ribonucleotides promote global B → A transitions, we also performed molecular modeling analyses to evaluate the structural consequences of higher ribonucleotide substitution levels. Increasing the ribonucleotide content increased the minor groove width toward values more similar to that of A-DNA, but even 50% ribonucleotide substitution did not fully convert the B-DNA to A-DNA. Comparing our structure with the structure of an RNase H2-bound DNA supports the conclusion that, as with other DNA-protein complexes, the DNA conformation is strongly influenced by the interaction with the protein.
核苷酸在复制过程中经常被整合到 DNA 中。它们被几种细胞酶识别和处理，它们在酵母核基因组中的持续存在会导致复制应激和基因组不稳定。因此，了解孤立核苷酸掺入对 DNA 结构的影响很重要。基于这一目标，我们描述了含有两个对称位置核糖核苷酸的自互补 Dickerson 十二聚体序列 [d(CGC)rGd(AATTCGCG)]² 的核磁共振结构。没有可观察到的 H(1)-H(2)标量耦合相互作用表明核糖采用 C3'-endo 构象。由于存在核苷酸引起的长程结构扰动仅限于相邻和跨螺旋核苷酸，而保持了全局 B 型 DNA 结构。由于晶体学研究表明孤立的核苷酸促进全局 B → A 转变，我们还进行了分子建模分析，以评估更高核苷酸取代水平的结构后果。增加核苷酸含量会使小沟宽度朝着更类似于 A-DNA 的值增加，但即使 50%的核苷酸取代也不能完全将 B-DNA 转化为 A-DNA。将我们的结构与 RNase H2 结合的 DNA 结构进行比较，支持这样的结论，即与其他 DNA-蛋白质复合物一样，DNA 构象受与蛋白质相互作用的强烈影响。
