Spontaneous DNA Lesions Modulate DNA Structural Transitions Occurring at Nuclease Hypersensitive Element III₁ of the Human c-myc Proto-Oncogene

Abstract

G quadruplex (G4) DNA is a noncanonical four-stranded DNA structure that can form in G repeats by stacking of planar arrays of four hydrogen-bonded guanines called G quartets, in the presence of potassium ions. In addition to a presumed function in the regulation of gene expression, G4 DNA also localizes to regions often characterized by genomic instability. This suggests that formation of this structure may interfere with DNA transactions, including processing of DNA damage at these sites. Here we have studied the effect of two spontaneous DNA lesions, the abasic site and 8-oxoguanine, on the transition from duplex to quadruplex DNA structure occurring at nuclease hypersensitive element III(1) (NHEIII(1)) of the human c-myc promoter. We show by dimethyl sulfate footprinting and RNA polymerase arrest assays that at physiological concentrations of potassium ions NHEIII(1) folds into two coexisting G4 DNA structures, myc-1245 and myc-2345, depending on which G runs are utilized for G quartet formation. We found that a single substitution of G12 of NHEIII(1) with a single abasic site or a single 8-oxoguanine prevented formation of G4 structure myc-2345 in favor of structure myc-1245, where the lesion was accommodated in a DNA loop formed by G11-AP12/(or 8-oxoG12)-G13-G14. Surprisingly, when an additional G to A base substitution was introduced at position 3 of NHEIII(1), we observed formation of myc-2345. The extent of this structural transition was modulated by the location and type of lesion within the G11-G14 repeat. Our data indicate that spontaneous lesions formed in the G4-forming sequence of c-myc NHEIII(1) affect the structural transitions occurring at this regulatory site, potentially altering transcription factor binding and DNA repair of lesions formed in this highly regulated sequence.

Human AP endonuclease inefficiently removes abasic sites within G4 structures compared to duplex DNA

“…To verify structure formation in the damaged-containing substrate, we have utilized FRET-based assays (38). Based on our recent findings (24) showing that the presence of an AP site at position 12 of the c-myc repeat generated G4 structure myc-1245, we modified the DNA strand containing the G4 sequence by incorporating two fluorophores, Cy3 at position 10 and Cy5 at position 28 of the c-myc G repeat during oligonucleotide synthesis (Figure 3A and B). Based on the myc-1245 conformation, when the G repeat sequence exists in the B-DNA conformation, the donor Cy3 (D) is relatively far from the acceptor Cy5 (A) (about 60 Å assuming a rise of about 3.3 Å per base pair) and will not be efficiently quenched by the acceptor (Figure 3A and B).…”

“…When the G4 DNA structure is formed, the donor Cy3 moves closer to the acceptor Cy5 and this decreases/quenches the donor fluorescence and increases the acceptor fluorescence due to resonance energy transfer (Figure 3A). First, we tested the FRET approach on a single-stranded DNA, ss c-myc-A12 substrate (Figure 3B, Table 1), which we had previously shown forms a stable c-myc-1245 G4 structure (24). As predicted, transition to G4 structure in the presence of KCl increased Cy5 acceptor emission compared to that measured for the untreated control.…”

“…Synthetic single-stranded DNA templates were prepared as previously described (24). The oligomer sequences are listed in Table 1.…”

“…We have recently developed novel approaches to promote the transition to G4 DNA under conditions that mimic those found in cells (23,24). In addition, we have shown that the presence of the most frequent spontaneous DNA lesions, abasic site and 8-oxoguanine within the G4 -forming sequence modulates the structural transition to G4 DNA in the nuclear hypersensitive element III 1 (NHEIII 1 ) from the c-myc gene (24).…”

“…In addition, we have shown that the presence of the most frequent spontaneous DNA lesions, abasic site and 8-oxoguanine within the G4 -forming sequence modulates the structural transition to G4 DNA in the nuclear hypersensitive element III 1 (NHEIII 1 ) from the c-myc gene (24). This G4-forming sequence is composed of a polypurine/polypyrimidine tract that can transition from duplex to quadruplex DNA structure in vivo (25) and has been implicated in c-myc gene regulation in normal and cancer cells (Figure 1A).…”