Direct observation of nucleic acid binding dynamics by the telomerase essential N-terminal domain

Nucleic Acids Research
Shankar ShastryMichael D Stone

Abstract

Telomerase is a specialized enzyme that maintains telomere length by adding DNA repeats to chromosome ends. The catalytic protein subunit of telomerase utilizes the integral telomerase RNA to direct telomere DNA synthesis. The telomerase essential N-terminal (TEN) domain is required for enzyme function; however, the precise mechanism of the TEN domain during catalysis is not known. We report a single-molecule study of dynamic TEN-induced conformational changes in its nucleic acid substrates. The TEN domain from the yeast Candida parapsilosis (Cp) exhibits a strong binding preference for double-stranded nucleic acids, with particularly high affinity for an RNA-DNA hybrid mimicking the template-product complex. Surprisingly, the telomere DNA repeat sequence from C. parapsilosis forms a DNA hairpin that also binds CpTEN with high affinity. Mutations to several residues in a putative nucleic acid-binding patch of CpTEN significantly reduced its affinity to the RNA-DNA hybrid and telomere DNA hairpin. Substitution of comparable residues in the related Candida albicans TEN domain caused telomere maintenance defects in vivo and decreased primer extension activity in vitro. Collectively, our results support a working model in which dyn...Continue Reading

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Citations

Sep 5, 2019·Cold Spring Harbor Perspectives in Biology·Linnea I Jansson, Michael D Stone
Aug 28, 2019·Cold Spring Harbor Perspectives in Biology·Yaqiang WangJuli Feigon
Feb 19, 2020·Nature Chemical Biology·Eric M PatrickJens C Schmidt
Nov 25, 2020·Proceedings of the National Academy of Sciences of the United States of America·Yaqiang WangJuli Feigon
Feb 3, 2021·Chemical Communications : Chem Comm·Ruiyuan ZhangXiaowen Xu

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Methods Mentioned

BETA
cross-linking studies
crosslinking studies
RAP
electrophoresis
in vitro transcription
FRET
electrophoretic mobility shift
nuclear
X-ray

Software Mentioned

HaMMY
smFRET
Prism
TASSER
GraphPad

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