Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

Abstract

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.oncanonical intramolecular structures of nucleic acids, such as a triplex and a quadruplex, are stabilized under conditions that mimic the crowded cellular conditions (1), and have been detected in cells (2, 3). In vitro and in vivo, guaninequadruplex (G-quadruplex or G4) formation inhibits transcription and translation of template nucleic acids (4-8). It is possible that the noncanonical structures act as "functional codes" triggered by different molecular environments, which regulate gene expression epigenetically (6, 7). As sequences capable of forming the noncanonical structures are found in telomeres and promoter regions of known oncogenes, alterations of the noncanonical structures could play important roles in the progression of cancer and in other diseases (9).One of the remarkable features of noncanonical structures of nucleic acids is the diversity of topologies. In the case of G4s, antiparallel, mixed, and parallel type structures have been characterized ( Fig. 1 A-C). The sequences complementary to regions capable of G4 formation are composed of tandem repeats of cytosine, and these C-rich regions can form a different type of tetraplex topology, which is the i-motif (10, 11). An intramolecular i-motif is formed upon the interaction of four C-rich regions. The structure has three loops and two parallel-stranded hairpin-like units stabilized by cytosine and protonated cytosine (C:C + ) base pairs that are vertically intercalated antiparallel to one another ( Fig. 1 D and E). The i-motif structures are categorized into class I and class II based on the lengths of the loops. C-rich sequences are found in telomeres and in the promoter regions of about 40% of human genes (12, 13). As the i-motif is stabilized by hydrogen bonding in C:C + base pairs (14, 15), acidic conditions stabilize the structure. As the i-motif can mediate transcriptional regulation of B-cell lymphoma 2 (Bcl2) oncogene in cells (16), the environment inside these cells might be favorable to i-motif formation....

Preferential targeting cancer-related i-motif DNAs by the plant flavonol fisetin for theranostics applications

“…From the therapeutic view point, we investigated whether the structural transformation by Fis influenced the replication reaction. Previously, we reported that the replication reaction can be affected by the formation of non-canonical structures on the template DNA and that the i-motif especially blocked the processivity of DNA polymerase efficiently 10 . For the replication analysis, a fluorescein-labelled primer and template DNA containing VEGF IM was designed as reported previously 10 .…”

Evaluation of the Stability of DNA i‐Motifs in the Nuclei of Living Mammalian Cells

“…Only recently,t he i-motifs in promoter and telomeric regions were proposed to be implicated in the regulation of transcription [3] and the integrity of telomeric DNA. [4] However,d espite all available indications of i-motif function in vivo,t he biological relevance of i-motifs has remained am atter of dispute for the following reasons:1 )The lack of direct experimental evidence for the existence of i-motifs in vivo and 2) the very strong dependence of i-motif stability on chemical and physical environmental factors such as pH that generally appear to be incompatible with the parameters of the intracellular space.W hile i-motifs are rather stable in acidic pH, low ionic strength, and dehydrating conditions,the propensity for i-motifs is generally lower under simulated physiological conditions comprising relatively high ionic strength (ca.…”

“…[12] In biological terms,o ur results support the concept that i-motif structures may act as active regulators of genomic DNAa nd potential drug targets. [3,4,13] …”

Advances in the cellular structural biology of nucleic acids

“…DNA i‐motifs are four‐stranded structures consisting of two parallel DNA duplexes zipped together by the intercalation of protonated cytosine–cytosine (C.CH + ) base pairs . These i‐motifs have recently been postulated to be actively involved in the regulation of DNA transcription . However, because i‐motif formation was never directly observed in vivo and because the formation of DNA i‐motifs is promoted by low ionic strength and acidic pH , that is, factors that are rather inconsistent with the physiological cellular environment, the existence of i‐motifs in vivo has been controversial.…”