Emerging Accessibility Patterns in Long Telomeric Overhangs

Abstract

We present single molecule experimental and computational modeling studies investigating the accessibility and folding landscape of human telomeric overhangs of physiologically relevant lengths. The overhangs contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate 1-7 tandem G-quadruplex (GQ) structures. Using FRET-PAINT, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to unfolded sites. We report accessibility patterns that periodically change with overhang length and provide insights about the underlying folding frustration. Overhangs that have 4n G-Tracts, (12, 16…), demonstrate maximum frustration, while those with 4n+2 G-Tracts, (14, 18…), have minimal frustration. We also developed a computational model that suggests positive folding cooperativity between neighboring GQs is required for persistence of such patterns. Our experimental and computational studies suggest lower folding stability at the junction between single and double stranded telomeric DNA, which has implications for Shelterin complex formation.

Impact of Shelterin Complex on Telomere Accessibility

“…The observed FRET levels are correlated with the position of the accessible G-Tracts with respect to the ssDNA/dsDNA junction, where Cy3 is located. We previously established the detection sensitivity and applicability of FRET-PAINT method by studying long telomeric sequences 39 . In the current study, we performed similar measurements to establish applicability of the method in the presence of POT1, which extends the overhang upon binding to it.…”

“…However, we observed an additional 2-fold reduction in the presence of shelterin compared to POT1-only case, which we propose to be due to reduced accessibility of the region between dsTEL and ssTEL in the presence of shelterin. This region of the overhang was demonstrated to be the most accessible region of ssTEL 39 and a potential site for the connection between POT1 and TRF1. Therefore, binding of shelterin to this junction region might be an effective way to enable a robust protection of ssTEL.…”

“…Both POT1 and shelterin reduced accessibility of Cy5-PNA although shelterin provided more efficient protection and resulted in more significant changes in Cy5-PNA accessibility maps. These findings suggest coordination between the components of shelterin that are localized on dsTEL and ssTEL play a role in reducing accessibility of the telomeric overhangs 39 .…”

“…In this assay, transient bindings of Cy5-labeled short peptide nucleic acid (PNA) strand to accessible regions of Cy3-labeled telomeric DNA are used to characterize the accessibility of the overhang. We previously showed that we can detect binding events of Cy5-PNA to ssTEL as long as ~168 nt, presumably because ssTEL becomes highly compact upon tandem GQ formation 39 . Due to their much higher stability, telomeric GQs largely inhibit binding of the short Cy5-PNA strand, which is complementary to only 7-nt of ssTEL.…”

“…To illustrate, the Cy5-PNA binding frequency for an overhang that contains 4 GGGTTA repeats (which can fold into a GQ) was an order of magnitude smaller than the binding frequency for an overhang that contains a single GGGTTA repeat (cannot form a GQ), even though it contains four times as many potential binding sites. Therefore, the large majority of GQs remain folded while PNA primarily binds to sites not protected within a GQ 39 .…”