POT1-TPP1 Regulates Telomeric Overhang Structural Dynamics

Hwang, Helen; Buncher, Noah; Opresko, Patricia L.; Myong, Sua

doi:10.1016/j.str.2012.08.018

Cited by 121 publications

(143 citation statements)

References 27 publications

(46 reference statements)

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“…Activity measurements of single telomerase molecules in the presence and absence of POT1-TPP1 demonstrated that POT1-TPP1 increased the rate of product formation and processivity of telomerase and triggered a more rapid dissociation from the telomeric primer (Hwang et al 2014). An important assumption underlying this study is that POT1-TPP1 is capable of sliding on ssDNA, an interesting hypothesis that awaits conclusive experimental verification (Hwang et al 2012). The importance of processivity stimulation by POT1-TPP1 in vivo is difficult to test because telomerase fails to localize to telomeres if the interaction between TPP1 and telomerase is lost.…”

Section: Telomerase Activation and Telomere Length Regulationmentioning

confidence: 99%

Human telomerase: biogenesis, trafficking, recruitment, and activation

2015

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Telomerase is the ribonucleoprotein enzyme that catalyzes the extension of telomeric DNA in eukaryotes. Recent work has begun to reveal key aspects of the assembly of the human telomerase complex, its intracellular trafficking involving Cajal bodies, and its recruitment to telomeres. Once telomerase has been recruited to the telomere, it appears to undergo a separate activation step, which may include an increase in its repeat addition processivity. This review covers human telomerase biogenesis, trafficking, and activation, comparing key aspects with the analogous events in other species.

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Section: Telomerase Activation and Telomere Length Regulationmentioning

confidence: 99%

Human telomerase: biogenesis, trafficking, recruitment, and activation

2015

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“…Similarly, RPA can bind very tightly to an ssDNA and dissociate rapidly when other SSBs are present in solution (20). Unlike studies (10,15,16,19) that were performed on an infinitely dilute solution, the findings of the study in ref. 20 apply to a wide range of conditions.…”

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confidence: 95%

“…For example, the complex between the protection of telomeres protein 1 (Pot1) and thiamine pyrophosphate protein 1 (TPP1; i.e., the Pot1-TPP1 complex) slides back and forth on telomeric DNA (19,20). Similarly, RPA can bind very tightly to an ssDNA and dissociate rapidly when other SSBs are present in solution (20).…”

mentioning

confidence: 99%

Molecular determinants of the interactions between proteins and ssDNA

Mishra

Levy

2015

Proc. Natl. Acad. Sci. U.S.A.

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ssDNA binding proteins (SSBs) protect ssDNA from chemical and enzymatic assault that can derail DNA processing machinery. Complexes between SSBs and ssDNA are often highly stable, but predicting their structures is challenging, mostly because of the inherent flexibility of ssDNA and the geometric and energetic complexity of the interfaces that it forms. Here, we report a newly developed coarse-grained model to predict the structure of SSBssDNA complexes. The model is successfully applied to predict the binding modes of six SSBs with ssDNA strands of lengths of 6-65 nt. In addition to charge-charge interactions (which are often central to governing protein interactions with nucleic acids by means of electrostatic complementarity), an essential energetic term to predict SSB-ssDNA complexes is the interactions between aromatic residues and DNA bases. For some systems, flexibility is required from not only the ssDNA but also, the SSB to allow it to undergo conformational changes and the penetration of the ssDNA into its binding pocket. The association mechanisms can be quite varied, and in several cases, they involve the ssDNA sliding along the protein surface. The binding mechanism suggests that coarse-grained models are appropriate to study the motion of SSBs along ssDNA, which is expected to be central to the function carried out by the SSBs.ssDNA | protein-DNA interaction | coarse-grained model

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“…3,4 It has also been found that telomere end-binding proteins control the folding and unfolding of DNA G-quadruplexes in vitro 5 and in vivo. 4 Telomeres are transcribed from the subtelomeric regions towards chromosome ends into telomeric repeat-containing RNA (TERRA).…”

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Mechanical unfolding of long human telomeric RNA (TERRA)

et al. 2013

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We report the first single molecule investigation of TERRA molecules.By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.Telomeres are nucleoprotein structures that protect chromosome ends from being recognized as DNA breaks.1 Mammalian telomere DNA consists of long tandem arrays of double-stranded TTAGGG repeats that are maintained by the telomerase. 2 The 3 0 end of each telomere terminates in a G-rich single-stranded overhang (150-200 nucleotides) that is able to self-fold into G-quadruplexes. The demonstration of the in vivo presence of DNA G-quadruplexes at telomeres suggests that these non-canonical secondary structures have a role in telomere end-protection, and therefore in chromosome stability. 3,4 It has also been found that telomere end-binding proteins control the folding and unfolding of DNA G-quadruplexes in vitro 5 and in vivo. 4Telomeres are transcribed from the subtelomeric regions towards chromosome ends into telomeric repeat-containing RNA (TERRA). 6,7 These non-coding RNA molecules contain subtelomere-derived sequences and an average of 34 GGGUUA repeats at their 3 0 end.8 TERRA acts as a scaffold for the assembly of telomeric proteins involved in telomere maintenance and telomeric heterochromatin formation. 9,10 Importantly, there is also evidence for the presence of TERRA RNA G-quadruplexes in living cells. Here, we report the synthesis and purification of TERRA molecules up to 96nt (twice the largest molecule previously analyzed by NMR), a size similar to the endogenous nuclear TERRA 8 (see ESI †).We have also used optical-tweezers (OT) to study RNA molecules with different numbers of possible quadruplexes ranging from 1 (Q1) to 6 (Q6). CD spectra of the RNA sequence Q4 in 25 mM potassium phosphate pH 7 buffer showed a positive band at 260 nm and a negative band at 240 nm (Fig. 1a), the characteristic signature of parallel-stranded G-quadruplex conformations. This result is in agreement with previously reported observations showing that other telomeric RNA sequences are folded into parallel G-quadruplex structures both in sodium and potassium solutions. 12,13,[16][17][18][19][20] It is also important to mention that no band at 300 nm was observed for this long telomeric RNA as it was reported for 22-mer and 45-mer telomeric RNA in ammonium acetate buffer. 15 On the other hand, the normalized CD spectra of the bimolecular G-quadruplex formed by r(UAGGGUUAGGGU) and Q4 showed a similar shape and amplitude, which indicates that most of the Q4 molecules form four G-quadruplexes (Fig. 1b). 21Thermal stability of Q4 was also measured by recording CD melting curves. To test the influence of the potassium concentration on the thermal stability we performed the experiments in two different potassium-containing buffers (Fig. 1c). Melting temperatures of 65.5 1C and 72.5 1C were obtained in 2...

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POT1-TPP1 Regulates Telomeric Overhang Structural Dynamics

Cited by 121 publications

References 27 publications

Human telomerase: biogenesis, trafficking, recruitment, and activation

Human telomerase: biogenesis, trafficking, recruitment, and activation

Molecular determinants of the interactions between proteins and ssDNA

Mechanical unfolding of long human telomeric RNA (TERRA)

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