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

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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.