Spontaneous DNA lesions modulate DNA structural transitions occurring at nuclease hypersensitive element III(1) of the human c-myc proto-oncogene.
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.
Department of Anatomy and Cell Biology, University of Florida College of Medicine, 1600 Southwest Archer Road, Gainesville, FL 32610, USA.
Joshua
J
Beckett
Jacob
J
Burns
Christopher
C
Broxson
Silvia
S
Tornaletti
D017123:Bacteriophage T7 / Q000201:enzymology; D004247:DNA / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D054856:G-Quadruplexes; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D012321:DNA-Directed RNA Polymerases