An intramolecular G-quadruplex structure is required for binding of telomeric repeat-containing RNA to the telomeric protein TRF2 - PubMed (original) (raw)

. 2012 Jul 25;134(29):11974-6.

doi: 10.1021/ja305734x. Epub 2012 Jul 13.

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An intramolecular G-quadruplex structure is required for binding of telomeric repeat-containing RNA to the telomeric protein TRF2

Giulia Biffi et al. J Am Chem Soc. 2012.

Free PMC article

Abstract

Telomeric repeat-containing RNA (TERRA) is important for telomere regulation, but the structural basis for how TERRA localizes to chromosome ends is unknown. Here we report on studies exploring whether the TERRA G-quadruplex structure is critical for binding to telomeres. We demonstrate that the telomeric protein TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than the TERRA sequence per se. We also show that TRF2 simultaneously binds TERRA and telomeric duplex or G-quadruplex DNA. These observations suggest that the TERRA G-quadruplex is a key feature of telomere organization.

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Figures

Figure 1

Figure 1

Biophysical analysis of TERRA oligonucleotides. Shown are CD spectra for TERRA-wt, TERRA-mut, and TERRA-lin oligonucleotides in the presence of 100 mM KCl. The TERRA-wt spectrum is characteristic of a parallel G-quadruplex.

Figure 2

Figure 2

UV spectroscopy for TERRA oligonucleotide characterization. (A) Thermal difference spectra of TERRA-wt, TERRA-mut, and TERRA-lin oligonucleotides in the presence of 10 mM KCl. The TERRA-wt spectrum shows a negative peak at 295 nm typical of G-quadruplex structures. (B) UV melting curve of TERRA-wt in the presence of 10 mM KCl. The calculated melting temperature (_T_m) is indicated.

Figure 3

Figure 3

TRF2 binds to TERRA via a G-quadruplex-dependent interaction. TRF2 binding curves for the TERRA-wt, TERRA-mut, and TERRA-lin oligonucleotides are shown, and the dissociation constant (_K_d) for TERRA-wt is indicated.

Figure 4

Figure 4

TRF2 binds more tightly to a telomeric DNA G-quadruplex structure than to a duplex, as shown by TRF2 binding curves for hTELO and hTELO-dup oligonucleotides. The corresponding _K_d values are indicated.

Figure 5

Figure 5

TRF2 binds to a hybrid telomeric DNA–TERRA G-quadruplex structure, as shown by the TRF2 binding curve for hTELO-hyb oligonucleotide. The _K_d value is indicated.

Figure 6

Figure 6

The TERRA RNA G-quadruplex and telomeric duplex DNA bind simultaneously to TRF2. A binding curve showing that TERRA-wt forms a complex with TRF2 bound to hTELO-dup as assessed by an ELISA assay is presented. Controls without added TRF2 are also shown. The diagram depicts the different components used.

Figure 7

Figure 7

The TERRA and telomeric DNA G-quadruplexes bind simultaneously to TRF2. A binding curve showing that TERRA-wt forms a complex with TRF2 bound to hTELO as assessed by an ELISA assay is presented. Controls without added TRF2 are also shown. The diagram depicts the different components used.

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