Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

Abstract

Author contributions: J.M., T.T.M.N., and T.H. designed research; J.M., M.A.M., and A.T. performed research; Y.R.C. contributed new reagents/analytic tools; J.M. and M.A.M. analyzed data; and J.M., M.A.M., Y.R.C., and T.H. wrote the paper.The authors declare no conflict of interest.

Streamlining effects of extra telomeric repeat on telomere folding revealed by fluorescence-force spectroscopy

“…Using smFRET and its combination with optical tweezers, which we refer to as fluorescence-force spectroscopy, we found evidence for preferential formation of GQs at the 3' end of five-repeat telomeric DNA, with GQs formed at the 5' end or at an internal position representing minority populations. Such GQs formed at the 3' end unravel non-cooperatively via strand slippage, contrary to cooperative unfolding of canonical GQs formed from four telomeric repeats (30). Furthermore, using a superhelicase-based vectorial folding assay, we show that a GQ form preferentially at the 3' end even when it is allowed to fold starting from the 5' side.…”

“…Dynamic exchange between GQ conformations in telomeric DNA spanning five to seven T2AG3 repeats have been observed using single molecule fluorescence resonance energy transfer (smFRET) (32) and single molecule optical tweezers studies of human telomeric DNA of four to seven repeats indicated that GQs more frequently form using four consecutive repeats (25). In a recent study we demonstrated extreme mechanical diversity heterogeneity of GQs in 22 nt long human telomeric DNA (four telomeric repeats, where at least six different mechanically distinct species were observed (30)). However, diversity in mechanical responses of GQs formed in longer telomeric DNA is yet to be explored.…”

“…In order to shed light on the conformational landscape, mutations can be introduced in biomolecules to selectively depopulate certain species from a heterogeneous population (24,29,40). Previous studies have also shown that guanine to thymine substitution of the central G of a hexanucleotide repeat T2AG3 precludes its participation in GQ formation in 100 mM K + or Na + (24,30) CD spectra of the hTel28 variants in 100 mM K + showed signatures of hybrid GQs as was the case for hTel28 itself (Fig. 1a) (39 variants were very stable as evidenced by steady E values ( Fig.…”

“…GQ formation requires a minimum of four G-rich segments (30). As human telomeric DNA contains multiple hexanucleotide (T2AG3) repeats and stretches for several kilobases, it has been assumed that GQs can form anywhere along the G-rich strand (31).…”

“…In this study, we employed smFRET to probe GQ formation of human telomeric DNA, spanning five and six T2AG3 repeats. Using guanine to thymine (G to T) mutation at the central G of a T2AG3 repeat, which is known to restrict the participation of the repeat in GQ formation (24,30), we created variants without positional multiplicity to help interpret the data. Using smFRET and its combination with optical tweezers, which we refer to as fluorescence-force spectroscopy, we found evidence for preferential formation of GQs at the 3' end of five-repeat telomeric DNA, with GQs formed at the 5' end or at an internal position representing minority populations.…”