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HRAS is silenced by two neighboring G-quadruplexes and activated by MAZ, a zinc-finger transcription factor with DNA unfolding property.

Cogoi S, Shchekotikhin AE, Xodo LE - Nucleic Acids Res. (2014)

Bottom Line: We have previously found that these G-quadruplexes bind to the zinc-finger transcription factors MAZ and Sp1.We also discovered that the two G-quadruplexes are strong targets for small anticancer molecules.In contrast, when one of the two G-quadruplexes was abrogated by point mutations, ATPD-1 repressed transcription by only 50%.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Biological Sciences, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy.

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(A) Sequence of hras-1 and densitometric scans of DMS-footprintings in 100 mM CsCl and 140 mM KCl; (B) Sequence of hras-2 and densitometric scans of DMS-footprinting in 100 mM CsCl and 140 mM KCl; (C) CD spectra of hras-1 in 50 mM Tris-HCl pH 7.4, 100 mM KCl, T = 20, 30, 40, 50, 55, 60, 65, 70, 80 and 90°C; (D) CD spectra of hras-2 in 50 mM Tris-HCl pH 7.5, 100 mM KCl, T = 20, 30, 40, 50, 55, 60, 65, 70, 80 and 90°C.
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Figure 2: (A) Sequence of hras-1 and densitometric scans of DMS-footprintings in 100 mM CsCl and 140 mM KCl; (B) Sequence of hras-2 and densitometric scans of DMS-footprinting in 100 mM CsCl and 140 mM KCl; (C) CD spectra of hras-1 in 50 mM Tris-HCl pH 7.4, 100 mM KCl, T = 20, 30, 40, 50, 55, 60, 65, 70, 80 and 90°C; (D) CD spectra of hras-2 in 50 mM Tris-HCl pH 7.5, 100 mM KCl, T = 20, 30, 40, 50, 55, 60, 65, 70, 80 and 90°C.

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).


HRAS is silenced by two neighboring G-quadruplexes and activated by MAZ, a zinc-finger transcription factor with DNA unfolding property.

Cogoi S, Shchekotikhin AE, Xodo LE - Nucleic Acids Res. (2014)

(A) Sequence of hras-1 and densitometric scans of DMS-footprintings in 100 mM CsCl and 140 mM KCl; (B) Sequence of hras-2 and densitometric scans of DMS-footprinting in 100 mM CsCl and 140 mM KCl; (C) CD spectra of hras-1 in 50 mM Tris-HCl pH 7.4, 100 mM KCl, T = 20, 30, 40, 50, 55, 60, 65, 70, 80 and 90°C; (D) CD spectra of hras-2 in 50 mM Tris-HCl pH 7.5, 100 mM KCl, T = 20, 30, 40, 50, 55, 60, 65, 70, 80 and 90°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: (A) Sequence of hras-1 and densitometric scans of DMS-footprintings in 100 mM CsCl and 140 mM KCl; (B) Sequence of hras-2 and densitometric scans of DMS-footprinting in 100 mM CsCl and 140 mM KCl; (C) CD spectra of hras-1 in 50 mM Tris-HCl pH 7.4, 100 mM KCl, T = 20, 30, 40, 50, 55, 60, 65, 70, 80 and 90°C; (D) CD spectra of hras-2 in 50 mM Tris-HCl pH 7.5, 100 mM KCl, T = 20, 30, 40, 50, 55, 60, 65, 70, 80 and 90°C.
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).

Bottom Line: We have previously found that these G-quadruplexes bind to the zinc-finger transcription factors MAZ and Sp1.We also discovered that the two G-quadruplexes are strong targets for small anticancer molecules.In contrast, when one of the two G-quadruplexes was abrogated by point mutations, ATPD-1 repressed transcription by only 50%.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Biological Sciences, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy.

Show MeSH
Related in: MedlinePlus