Emerging accessibility patterns in long telomeric overhangs.

We present single-molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate one to seven tandem G-quadruplex (GQ) structures. Using the FRET-PAINT method, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [4n]G-Tracts, (12, 16, 20…) demonstrate the broadest accessibility patterns where the peptide nucleic acid probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22…) have narrower patterns where the neighborhood of the junction between single- and double-stranded telomeres is most accessible. We interpret these results as the folding frustration being higher in [4n]G-Tract constructs compared to [4n+2]G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of the junction between single- and double-stranded telomeres is least stable and most accessible, which is significant as this is a potential site where the connection between POT1/TPP1 (bound to single-stranded telomere) and other shelterin proteins (localized on double-stranded telomere) is established.

Shelterin reduces the accessibility of telomeric overhangs.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

Detecting secondary structure formation with FRET-PAINT.

We present single molecule studies demonstrating the capabilities of the FRET-PAINT method to detect secondary structures that would be challenging to detect with alternative methods, particularly single molecule FRET (smFRET). Instead of relying on the change in end-to-end separation as in smFRET, we use the change in accessibility to a small probe as the criterion for secondary structure formation and relative stability. As a model system, we study G-triplex formation by human telomeric repeat sequences in different structural contexts.

Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction.

Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24-144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3'- or 5'-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.

Structure, Topology, and Stability of Multiple G-quadruplexes in Long Telomeric Overhangs.

Telomeres and their single stranded overhangs gradually shorten with successive cell divisions, as part of the natural aging process, but can be elongated by telomerase, a nucleoprotein complex which is activated in the majority of cancers. This prominent implication in cancer and aging has made the repetitive telomeric sequences (TTAGGG repeats) and the G-quadruplex structures that form in their overhangs the focus of intense research in the past several decades. However, until recently most in vitro efforts to understand the structure, stability, dynamics, and interactions of telomeric overhangs had been focused on short sequences that are not representative of longer sequences encountered in a physiological setting. In this review, we will provide a broad perspective about telomeres and associated factors, and introduce the agents and structural characteristics involved in organizing, maintaining, and protecting telomeric DNA. We will also present a summary of recent research performed on long telomeric sequences, nominally defined as those that can form two or more tandem G-quadruplexes, i.e., which contain eight or more TTAGGG repeats. Results of experimental studies using a broad array of experimental tools, in addition to recent computational efforts will be discussed, particularly in terms of their implications for the stability, folding topology, and compactness of the tandem G-quadruplexes that form in long telomeric overhangs.

Stalling of Transcription by Putative G-quadruplex Sequences and CRISPR-dCas9.

Putative G-quadruplex forming sequences (PQS) have been identified in promoter sequences of prominent genes that are implicated among others in cancer and neurological disorders. We explored mechanistic aspects of CRISPR-dCas9-mediated gene expression regulation, which is transient and sequence specific unlike alternative approaches that lack such specificity or create permanent mutations, using the PQS in tyrosine hydroxylase (TH) and c-Myc promoters as model systems. We performed in vitro ensemble and single molecule investigations to study whether G-quadruplex (GQ) structures or dCas9 impede T7 RNA polymerase (RNAP) elongation process and whether orientation of these factors is significant. Our results demonstrate that dCas9 is more likely to block RNAP progression when the non-template strand is targeted. While the GQ in TH promoter was effectively destabilized when the dCas9 target site partially overlapped with the PQS, the c-Myc GQ remained folded and stalled RNAP elongation. We also determined that a minimum separation between the transcription start site and the dCas9 target site is required for effective stalling of RNAP by dCas9. Our study provides significant insights about the factors that impact dCas9-mediated transcription regulation when dCas9 targets the vicinity of sequences that form secondary structures and provides practical guidelines for designing guide RNA sequences.

A single molecule investigation of i-motif stability, folding intermediates, and potential as in-situ pH sensor.

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for low pH levels (pH < 6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Förster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the intermediate states visited during i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor to establish the time scale for the pH drop in a commonly used oxygen scavenging system.

Rtt105 regulates RPA function by configurationally stapling the flexible domains.

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.