Investigation of Intercalated (I) Motif DNA Structure at Telomeric Cytosine-Rich Strand in Human Cells Genetically Altered for DNA Helicases

I-基序是一个富含胞嘧啶的DNA序列，它形成了一个非规范的DNA结构，不同于传统的化学键规则，该规则规定了DNA双螺旋的三维排列和严格的互补碱基对(腺嘌呤(A)：胸腺嘧啶(T)；鸟嘌呤(G)：胞嘧啶(C))和定义的碱基对堆积排列(Bp)。相反，I基序形成两个插入的平行链双链，通过半质子化的C-Cbps连接在一起。通过计算机算法Quadparser对人类基因组的分析确定了5125个DNA序列，这些DNA序列可以折叠成I基序结构(也称为C-四链或C4)。这些预计会形成C4的富碳序列大多存在于端粒、着丝粒和基因启动子区域。 基因组I基序DNA结构的调节是调节基因表达或端粒代谢的潜在机制，可能与癌症治疗、细胞衰老和衰老有关。羧化的单壁碳纳米管(SWNTs)是第一个被发现选择性稳定人类端粒I基序DNA结构的配体，从而在体外和人类细胞中抑制端粒酶的端粒重复合成。单壁碳纳米管稳定I基序导致端粒解帽，并诱导端粒DNA损伤反应，导致细胞周期停滞、衰老或凋亡。最近，(在NCI多样性集中)发现了专门结合I基序DNA结构并使其稳定的小分子(NSC 1389484；NSC 3098745)或与富含C的发夹结合，从而阻止C4的形成(NSC 592764；NSC 1463976)。虽然这些化合物被测试了它们对基因启动子活性的影响，但它们对端粒新陈代谢的影响要么没有提到，要么没有报道。 尽管实验证据表明C4DNA结构在体内形成并产生独特的生物学后果，但其分子代谢途径尚不清楚。例如，虽然我们和其他实验室的工作表明，专门的DNA解旋酶具有分解人类基因组中富含G的序列形成的G-四链(G4)DNA的重要功能，但还没有解旋酶与I-基序四链相互作用或催化解离的报道。此外，还没有研究详细说明I-基序DNA结构对体内复制的影响；然而，一项体外研究表明，大肠杆菌DNA聚合酶片段Klenow的DNA合成被I-基序DNA结构所阻碍。

The I-motif is a cytosine-rich DNA sequence that forms a non-canonical DNA structure deviating from the conventional chemical bonding rules that dictate the three-dimensional arrangement of the DNA double helix with strict complementary base pairing (adenine (A): thymine (T); guanine (G): cytosine (C)) and a defined stacking arrangement of the base pairs (bps). Instead, the I-motif forms two intercalated parallel-stranded duplexes held together by hemi-protonated C-C bps. Analysis of the human genome by the computer algorithm Quadparser identified 5,125 DNA sequences that can fold into I-motif structures (also designated C-quadruplex or C4). Most of these C-rich sequences predicted to form C4 are found in telomeres, centromeres, and gene promoter regions. Regulation of genomic I-motif DNA structure is a potential mechanism to modulate gene expression or telomere metabolism with implications for cancer therapy, cellular senescence and aging. Carboxylated single-walled carbon nanotubes (SWNTs) were the first ligand found to selectively stabilize human telomeric I-motif DNA structures, thereby inhibiting telomeric repeat synthesis by telomerase in vitro and in human cells. I-motif stabilization by SWNTs resulted in telomere uncapping and induced a telomeric DNA damage response that led to cell cycle arrest, senescence or apoptosis. Recently, small molecules (in the NCI Diversity Set) were discovered that specifically bind I-motif DNA structures and stabilize them (NSC 1389484 ; NSC 3098745) or bind to the C-rich hairpin, thereby preventing C4 formation (NSC 592764; NSC 1463976). While these compounds were tested for their effects on gene promoter activity, their impact on telomere metabolism was either not addressed or reported. Despite experimental evidence that C4 DNA structures form in vivo and exert unique biological consequences, their molecular metabolic pathways are poorly understood. For example, while work from our lab and others demonstrated that specialized DNA helicases have been characterized for their vital functions to resolve G-quadruplex (G4) DNA formed by G-rich sequences in the human genome, there are no reports that helicase enzymes interact with or catalytically unwind I-motif quadruplexes. Moreover, there have been no studies detailing the effects of I-motif DNA structures on replication in vivo; however, one in vitro study demonstrated that DNA synthesis by the E. coli DNA polymerase fragment Klenow was stalled by a I-motif DNA structure.