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

I-基序是富含胞嘧啶的DNA序列，其形成偏离常规化学键合规则的非规范DNA结构，所述常规化学键合规则规定具有严格互补碱基配对（腺嘌呤（A）：胸腺嘧啶（T）;鸟嘌呤（G）：胞嘧啶（C））的DNA双螺旋的三维排列和碱基对（bps）的限定堆叠排列。相反，I-基序形成两个插入的平行链双链体，通过半质子化的C-C bp保持在一起。通过计算机算法Quadparser对人类基因组的分析确定了5，125个可以折叠成I基序结构（也称为C-quadruplex或C4）的DNA序列。这些富含C的序列中的大多数被预测形成C4在端粒、着丝粒和基因启动子区域中被发现。 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，对癌症治疗、细胞衰老和衰老具有重要意义。羧化单壁碳纳米管（SWNTs）是第一个被发现选择性稳定人端粒I基序DNA结构的配体，从而抑制端粒酶在体外和人细胞中的端粒重复序列合成。单壁碳纳米管的I基序稳定导致端粒脱帽，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，发现小分子（在NCI多样性组中）特异性结合I基序DNA结构并稳定它们（NSC 1389484 ; NSC 3098745）或结合富含C的发夹，从而防止C4形成（NSC 592764; NSC 1463976）。虽然测试了这些化合物对基因启动子活性的影响，但它们对端粒代谢的影响没有得到解决或报道。 尽管有实验证据表明C4 DNA结构在体内形成并产生独特的生物学后果，但对其分子代谢途径知之甚少。例如，虽然我们实验室和其他人的工作表明，专门的DNA解旋酶已被表征为它们的重要功能，以解析由人类基因组中富含G的序列形成的G-四链体（G4）DNA，但没有报道解旋酶与I-基序四链体相互作用或催化解链。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，E.大肠杆菌DNA聚合酶片段Klenow被一个I基序DNA结构所阻断。

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.