Unraveling secrets of telomeres: one molecule at a time

Abstract

Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.

Keywords: Atomic force microscopy; FRET; Fluorescence microscopy; Laser tweezers; Magnetic tweezers; Protein–DNA interactions; Single-molecule imaging; Telomeres.

Fig. 1

Human telomeres. Top: Shelterin proteins, including TRF1, TRF2, TIN2, RAP1, TPP1, and POT1, interact with many proteins involved in cell cycle progression and DNA repair [14]. The telomerase complex is regulated by shelterin proteins and DNA structures at telomeres, such as T-loop and G-quadruplexes. Telomeric DNA can be modeled into a T-loop structure (bottom left) [25] or exists as extrachromosomal telomeric circles (t-circles, bottom right) [137]. Reprinted with permissions from [25] (1999 Elsevier) and [137] (2004 American Society for Microbiology).

Fig. 2

Single-molecule imaging techniques used in the studies of telomere structure and function. (A) Atomic force microscopy (AFM) (left) and force spectroscopy (right) setup [71].. (C) Single-molecule FRET setup [92].

Fig. 3

Single-molecule studies of G-quadruplex structures. (A) An AFM image of beads-on-a-string structures formed by (TTAGGG)₁₆ [52]. (B) High-speed AFM imaging of interstrand G-quadruplex structure (X-shape) formation in real-time using nanoscaffold [69]. Reprinted with permission from [69], 2010 American Chemical Society (C) laser tweezers. Left: a shematic representation of laser tweezers set up. Right: Dynamic force spectroscopy study of G-quadruplexes using laser tweezers. Top right: Force distributions are consistent with two quadruplex conformations, parallel (blue) and antiparallel (red). Bottom right: proposed unfolding model: 2–3 bp disruption of the G-quadruplex under force leads to the transition state and disruption of G-quadruplex structures [72]. Reprinted with permission from [72], 2012 American Physical Society. (D) magnetic tweezers and smFRET integration [73] for studying G-quadruplexes. Reprinted with permission from [73], 2013 Oxford University Press.

Fig. 4

Single-molecule studies of telomerase. (A) smFRET study of the structure of isolated PK (left) and full-length RNA sequence (right) [80]. The isolated PK sequence form stable PK structure indicated by high FRET. In contrast, the full-length sequences do not form stable PK structure indicated by low FRET. Reprinted with permission from [80], 2011 National Academy of Sciences. (B) Studying the dynamics of PK folding and unfolding using laser tweezers [83]. Top: the experimental setup, bottom: representative force-extension curves. Reprinted with permission from [83], 2007 RNA Society.

Fig. 5

Mechanisms of G-quadruplex unfolding mediated by POT1. (A) POT1 disrupts G-quadruplexes by acting as a “steric drive” to promote unfolding [52]. Left panel: a representative AFM surface plot of Tel16 DNA containing 16 TTAGGG repeats in the presence of POT1. The thick arrow points to a structure with folded G4. The triangle points to an individual POT1 molecule. The thin arrow points to a structure with multiple POT1 proteins. Right panel: cross-section of the molecule highlighted in the left panel by the dotted line demonstrating that G-quadruplex (left peak) and POT1 (right peak) coexist on the same molecule. (B) POT1 binds to single-stranded telomeric DNA resulted in a stepwise FRET decrease [92]. Left: Schematic of the experimental design. Middle: POT1 binding sites marked in gray. Right: the numbers of steps shown in the single-molecule FRET traces are consistent with two POT1 monomers bind the (TTAGGG)₄ sequence one OB fold at a time. Reprinted with permission from [92], 2012 Elsevier.

Fig. 6

TRF1 and TRF2 play important architectural roles at telomeres. (A) Schematic representations of the domain structures of TRF1 and TRF2. A: Acidic domain, B: Basic domain. M: Myb type domain. (B) An EM micrograph showing that TRF1 induces DNA looping between two AGGGTT sites separated by 205 bp (left). Right: models for how TRF1 uses one or two Myb domains to contact specific DNA sites [114]. (C) An EM micrograph showing that TRF1 promotes parallel pairing of telomeric tracts [51]. (D) An EM micrograph showing that TRF2 promotes T-loop formation in vitro [25]. (E) AFM imaging showing higher oligomers of TRF2 with larger volumes condensed DNA leading to shortening of DNA length [118]. Reprinted with permissions from [114] (1999 Nature Publishing Group), [51] (1998 Elsevier), [25] (1999 Elsevier), and [118] (2012 Oxford University Press).

Fig. 7

DNA tightrope assay based oblique-angle fluorescence imaging of TRF1- and TRF2-QDs on λ DNA tightropes [124]. (A) Schematic representations of TRF1- and TRF2-QD conjugates. (B) A schematic drawing of the DNA tightropes between silica beads; red ball: QD; proteins: green balls. The drawing is not to scale. (C) A schematic drawing of the ligated T270 DNA substrate (top) and a representative fluorescence image of dual color (655 and 565 nm) labeled TRF1-QDs on the ligated T270 DNA substrate. (D) TRF1 and TRF2 show different diffusional properties over telomeric region versus non-telomeric regions. Kymographical analysis of dual color (655 and 565 nm) labeled TRF1 (top) and TRF2 (bottom) on the T270 DNA with alternating telomeric (purple) and non-telomeric regions (blue). The scale bar is 1 µm. Reprinted with permission from [124], 2013 Oxford University Press.