Shiekh, S. | Search bioRxiv

bioRxiv2022

Impact of Shelterin Complex on Telomere Accessibility

Shelterin plays critical roles in maintaining and protecting telomeres by regulating access of various physiological agents to telomeric DNA. We present single molecule measurements investigating the impact of the POT1 and a four-component shelterin complex on the accessibility of human telomeric DNA overhangs with physiologically relevant lengths (28-150 nt), which to our knowledge is the first direct approach to measure this effect on such telomeric constructs. To quantify telomere accessibility, we monitored transient binding events of a short peptide nucleic acid (PNA) probe that is complementary to telomeric overhangs using FRET-PAINT. Although POT1 has a mild G-quadruplex unfolding activity, it reduced 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 reduced the accessibility of telomeric overhangs by [~]5-fold. This enhanced protection suggests shelterin restructures the region between single and double stranded telomere, which is otherwise the most accessible part of the overhang, by a synergistic cooperation of shelterin components located on single and double stranded telomere.

biophysics↗

bioRxiv2022

Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. 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 (RIPs). 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 RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

biochemistry↗

bioRxiv2021

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 1-7 tandem G-quadruplex (GQ) structures. Using 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 PNA 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 telomere 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 telomere 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. Significance StatementThe ends of eukaryotic linear chromosomes are capped by telomeres which terminate with a single-stranded overhang. Telomeric overhangs fold into compact structures, called G-quadruplex, that inhibit access to these critical genomic sites. We report single molecule measurements and computational modeling studies probing the accessibility of a set of human telomeric overhangs that covers a significant portion of the physiologically relevant length scale. We observe novel accessibility patterns which have a well-defined periodicity and show that certain regions are significantly more accessible than others. These accessibility patterns also suggest the underlying folding frustration of G-quadruplexes depends on telomere length. These patterns have significant implications for regulating the access of DNA processing enzymes and DNA binding proteins that can target telomeric overhangs.

biophysics↗