TY - JOUR
T1 - Telomeric overhang length determines structural dynamics and accessibility to telomerase and ALT-associated proteins
AU - Hwang, Helen
AU - Kreig, Alex
AU - Calvert, Jacob
AU - Lormand, Justin
AU - Kwon, Yongho
AU - Daley, James M.
AU - Sung, Patrick
AU - Opresko, Patricia L.
AU - Myong, Sua
N1 - Funding Information:
We thank Walter Chazin (Vanderbilt University) for generously providing recombinant human replication protein A (R01 GM65484), Thomas Cech (University of Colorado at Boulder) for the Flag-tagged telomerase overexpression plasmid, Noah Buncher for technical support on protein purification, Ankur Jain for his help with the telomerase pull-down assay, and Ramreddy Tippana for helpful discussions. Support for this work was provided by an NIH Director’s New Innovator Award (343 NIH 1 DP2 GM105453 A) and the American Cancer Society (Research Scholar grant RSG-12-066-01-DMC) to S.M., the Linda Su-Nan Chang Sah Doctoral Fellowship to H.H., NIH grant RO1 ES015632 to P.S., and NIH grant ES0515052 and the David Scaife Foundation to P.L.O.
PY - 2014/6/10
Y1 - 2014/6/10
N2 - Summary The G-rich single-stranded DNA at the 3′ end of human telomeres can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase or the recombination-based alternative lengthening of telomere (ALT) mechanism requires protein loading on the overhang. Using single-molecule fluorescence spectroscopy, we discovered that lengthening the telomeric overhang also increased the rate of dynamic exchanges between structural conformations. Overhangs with five to seven TTAGGG repeats, compared with four repeats, showed much greater dynamics and accessibility to telomerase binding and activity and loading of the ALT-associated proteins RAD51, WRN, and BLM. Although the eight repeats are highly dynamic, they can fold into two GQs, which limited protein accessibility. In contrast, the telomere-specific protein POT1 is unique in that it binds independently of repeat number. Our results suggest that the telomeric overhang length and dynamics may contribute to the regulation of telomere extension via telomerase action and the ALT mechanism.
AB - Summary The G-rich single-stranded DNA at the 3′ end of human telomeres can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase or the recombination-based alternative lengthening of telomere (ALT) mechanism requires protein loading on the overhang. Using single-molecule fluorescence spectroscopy, we discovered that lengthening the telomeric overhang also increased the rate of dynamic exchanges between structural conformations. Overhangs with five to seven TTAGGG repeats, compared with four repeats, showed much greater dynamics and accessibility to telomerase binding and activity and loading of the ALT-associated proteins RAD51, WRN, and BLM. Although the eight repeats are highly dynamic, they can fold into two GQs, which limited protein accessibility. In contrast, the telomere-specific protein POT1 is unique in that it binds independently of repeat number. Our results suggest that the telomeric overhang length and dynamics may contribute to the regulation of telomere extension via telomerase action and the ALT mechanism.
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U2 - 10.1016/j.str.2014.03.013
DO - 10.1016/j.str.2014.03.013
M3 - Article
C2 - 24836024
AN - SCOPUS:84902289206
VL - 22
SP - 842
EP - 853
JO - Structure with Folding & design
JF - Structure with Folding & design
SN - 0969-2126
IS - 6
ER -