Das Kohal, Srivastava Mrinal, Raghavan Sathees C
Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
PLoS One. 2016 Jul 14;11(7):e0158794. doi: 10.1371/journal.pone.0158794. eCollection 2016.
G-quadruplexes are one of the most commonly studied non-B DNA structures. Generally, these structures are formed using a minimum of 4, three guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In this study, guanines along with bulges, also referred to as GNG motifs have been extensively studied using recently reported HOX11 breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the contribution from continuous G-tracts may be dispensible during G-quadruplex formation when such motifs are flanked by GNGs. Importantly, the positioning and number of GNG/GNGNG can also influence the formation of G-quadruplexes. Further, we assessed three genomic regions from HIF1 alpha, VEGF and SHOX gene for G-quadruplex formation using GNG motifs. We show that HIF1 alpha sequence harbouring GNG motifs can fold into intramolecular G-quadruplex. In contrast, GNG motifs in mutant VEGF sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two continuous stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (SHOX), it can fold into an intramolecular G-quadruplex. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody, BG4 to SHOX G-quadruplex. Overall, our study provides novel insights into the role of GNG motifs in G-quadruplex structure formation which may have both physiological and pathological implications.
G-四链体是研究最为广泛的非B型DNA结构之一。一般来说，这些结构至少由4个鸟嘌呤序列形成，有3个鸟嘌呤束，连接环的长度从1到7个碱基不等。最近的研究报道了与这一普遍规律的偏差。其中一种偏差是鸟嘌呤束中存在凸起。在本研究中，以最近报道的HOX11断裂点脆性区域I为模型模板，对带有凸起的鸟嘌呤（也称为GNG基序）进行了广泛研究。通过策略性诱变方法，我们发现当这些基序两侧为GNG时，连续鸟嘌呤序列在G-四链体形成过程中的作用可能是可有可无的。重要的是，GNG/GNGNG的定位和数量也会影响G-四链体的形成。此外，我们利用GNG基序评估了来自HIF1α、VEGF和SHOX基因的三个基因组区域的G-四链体形成情况。我们发现含有GNG基序的HIF1α序列可以折叠成分子内G-四链体。相比之下，突变型VEGF序列中的GNG基序不能参与结构形成，这表明GNG的使用依赖于上下文。重要的是，我们发现当两个连续的鸟嘌呤序列在天然序列（SHOX）中两侧被两个独立的GNG基序包围时，它可以折叠成分子内G-四链体。最后，我们展示了G-四链体结合蛋白Nucleolin和G-四链体抗体BG4与SHOX G-四链体的特异性结合。总体而言，我们的研究为GNG基序在G-四链体结构形成中的作用提供了新的见解，这可能具有生理和病理意义。
