HRAS is silenced by two neighboring G-quadruplexes and activated by MAZ, a zinc-finger transcription factor with DNA unfolding property.
Bottom Line: We have previously found that these G-quadruplexes bind to the zinc-finger transcription factors MAZ and Sp1.In contrast, when one of the two G-quadruplexes was abrogated by point mutations, ATPD-1 repressed transcription by only 50%.Our study provides relevant information for the rationale design of targeted therapy drugs specific for the HRAS oncogene.
Affiliation: Department of Medical and Biological Sciences, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy.Show MeSH
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Mentions: As sequences hras-1 and hras-2 are composed of runs of guanines, they are inclined to fold into G-quadruplex structures (Figure 2A–D) (9). DMS-footprinting was used to determine that these guanines are involved in the formation of G-quadruplexes. All hras-1 guanines are reactive to DMS in 100 mM CsCl, while in 140 mM KCl only G8 and G14 are reactive. This unambiguously shows that G3–G5, G10–G12, G17–G19, G23–G25 are involved in the formation of the G-tetrad core of the quadruplex. Hras-1 shows a CD spectrum with positive and negative ellipticities at 295 and 256 nm, respectively, suggesting that the sequence folds into an antiparallel quadruplex. CD and FRET experiments as a function of temperature showed that hras-1 melts in a cooperative manner with a biphasic profile in 100 mM KCl, indicating that it folds into two quadruplexes with TM's of ∼53 and ∼65°C. Moreover, due to its moderate stability, quadruplex hras-1 is unable to arrest Taq polymerase in primer extension experiments, but it does so in the presence of a G4 stabilizer (phthalocyanine) (S2,a).
Affiliation: Department of Medical and Biological Sciences, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy.