Characterization of Physical and Functional Anchor Site Interactions in Human Telomerase

ABSTRACT Telomerase is a ribonucleoprotein reverse transcriptase (RT) that processively synthesizes telomeric repeats onto the ends of linear chromosomes to maintain genomic stability. It has been proposed that the N terminus of the telomerase protein subunit, telomerase RT (TERT), contains an anchor site that forms stable interactions with DNA to prevent enzyme-DNA dissociation during translocation and to promote realignment events that accompany each round of telomere synthesis. However, it is not known whether human TERT (hTERT) can directly interact with DNA in the absence of the telomerase RNA subunit. Here we use a novel primer binding assay to establish that hTERT forms stable and specific contacts with telomeric DNA in the absence of the human telomerase RNA component (hTR). We show that hTERT-mediated primer binding can be functionally uncoupled from telomerase-mediated primer extension. Our results demonstrate that the first 350 amino acids of hTERT have a critical role in regulating the strength and specificity of protein-DNA interactions, providing additional evidence that the TERT N terminus contains an anchor site. Furthermore, we establish that the RT domain of hTERT mediates important protein-DNA interactions. Collectively, these data suggest that hTERT contains distinct anchor regions that cooperate to help regulate telomerase-mediated DNA recognition and elongation.

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SURFACE PLASMON RESONANCE SPECTROSCOPY AND QUARTZ CRYSTAL MICROBALANCE STUDY OF PROTEIN-DNA INTERACTIONS IN HORMONE RECEPTOR BIOLOGY

COSMOS ◽

10.1142/s0219607709000415 ◽

2009 ◽

Vol 05 (01) ◽

pp. 79-95

Author(s):

XIAODI SU

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Quartz Crystal Microbalance ◽

Conformational Changes ◽

Plasmon Resonance ◽

Quartz Crystal ◽

Resonance Spectroscopy ◽

Dna Interactions ◽

Protein Dna Interactions

Surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) are surface sensitive analytical techniques capable of real-time monitoring of biomolecular interactions. In this article we review our past work on the use of these two techniques for studying protein–DNA interactions, exemplified with estrogen receptors (ER) and their response elements (ERE). Various assay schemes have been developed for a comprehensive characterization of ER–ERE interactions in terms of sequence specificity, binding affinity, stoichiometry, ligand effects on binding dynamics and conformational changes in the proteins and DNA. These are all important characteristics underlining the mechanism of ER-mediated gene transcription. With these studies we have made the following demonstrations to describe the advantages of these two techniques, namely (i) SPR technique is superior and more versatile than conventional (electrophoretic mobility shift assay) EMSA for studying protein-DNA interactions; (ii) QCM is an alternative tool for studying conformational changes in protein–DNA complexes and (iii) combinational SPR and QCM analysis provides additional characterization of biomolecular films, e.g. film thickness, water content, and conformation rigidity etc.

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An Anchor Site–Type Defect in Human Telomerase That Disrupts Telomere Length Maintenance and Cellular Immortalization

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0148 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3152-3161 ◽

Cited By ~ 37

Author(s):

Tara J. Moriarty ◽

Ryan J. Ward ◽

Michael A.S. Taboski ◽

Chantal Autexier

Keyword(s):

Telomerase Activity ◽

Eukaryotic Cell ◽

Telomeric Dna ◽

Interaction Domain ◽

C Terminus ◽

Human Telomerase ◽

Rna Interaction ◽

Dna Primers ◽

Reduced Activity

Telomerase-mediated telomeric DNA synthesis is important for eukaryotic cell immortality. Telomerase adds tracts of short telomeric repeats to DNA substrates using a unique repeat addition form of processivity. It has been proposed that repeat addition processivity is partly regulated by a telomerase reverse transcriptase (TERT)-dependent anchor site; however, anchor site-mediating residues have not been identified in any TERT. We report the characterization of an N-terminal human TERT (hTERT) RNA interaction domain 1 (RID1) mutation that caused telomerase activity defects consistent with disruption of a template-proximal anchor site, including reduced processivity on short telomeric primers and reduced activity on substrates with nontelomeric 5′ sequences, but not on primers with nontelomeric G-rich 5′ sequences. This mutation was located within a subregion of RID1 previously implicated in biological telomerase functions unrelated to catalytic activity (N-DAT domain). Other N-DAT and C-terminal DAT (C-DAT) mutants and a C-terminally tagged hTERT-HA variant were defective in elongating short telomeric primers, and catalytic phenotypes of DAT variants were partially or completely rescued by increasing concentrations of DNA primers. These observations imply that RID1 and the hTERT C terminus contribute to telomerase's affinity for its substrate, and that RID1 may form part of the human telomerase anchor site.

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Functional Characterization of DNA-binding Domains of the Subunits of the Heterodimeric Aryl Hydrocarbon Receptor Complex Imputing Novel and Canonical Basic Helix-Loop-Helix Protein-DNA Interactions

Journal of Biological Chemistry ◽

10.1074/jbc.271.15.8843 ◽

1996 ◽

Vol 271 (15) ◽

pp. 8843-8850 ◽

Cited By ~ 50

Author(s):

Steven G. Bacsi ◽

Oliver Hankinson

Keyword(s):

Functional Characterization ◽

Receptor Complex ◽

Dna Interactions ◽

Dna Binding Domains ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Aryl Hydrocarbon ◽

Binding Domains ◽

Protein Dna Interactions

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Combining conservation and species-specific differences to determine how human telomerase binds telomeres

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911912116 ◽

2019 ◽

Vol 116 (52) ◽

pp. 26505-26515 ◽

Cited By ~ 1

Author(s):

Valerie M. Tesmer ◽

Eric M. Smith ◽

Oana Danciu ◽

Shilpa Padmanaban ◽

Jayakrishnan Nandakumar

Keyword(s):

Structural Model ◽

Species Specificity ◽

Telomeric Dna ◽

Protein Subunit ◽

High Resolution Structure ◽

Human Telomerase ◽

Species Specific ◽

Interaction Surface ◽

Telomeric Protein ◽

Oligonucleotide Binding

Telomerase catalyzes telomeric DNA synthesis at chromosome ends to allow for continued cell division. The telomeric protein TPP1 is essential for enhancing the processivity of telomerase and recruiting the enzyme to telomeres. The telomerase interaction surface on human TPP1 has been mapped to 2 regions of the N-terminal oligosaccharide/oligonucleotide-binding (OB) domain, namely the TPP1 glutamate (E) and leucine (L)-rich (TEL) patch and the N terminus of TPP1-oligosaccharide/oligonucleotide-binding (NOB) region. To map the telomerase side of the interface, we exploited the predicted structural similarities for human andTetrahymena thermophilatelomerase as well as the species specificity of human and mouse telomerase for their cognate TPP1 partners. We show that swapping in the telomerase essential N-terminal (TEN) and insertions in fingers domain (IFD)-TRAP regions of the human telomerase catalytic protein subunit TERT into the mouse TERT backbone is sufficient to bias the species specificity toward human TPP1. Employing a structural homology-based mutagenesis screen focused on surface residues of the TEN and IFD regions, we identified TERT residues that are critical for contacting TPP1 but dispensable for other aspects of telomerase structure or function. We present a functionally validated structural model for how human telomerase engages TPP1 at telomeres, setting the stage for a high-resolution structure of this interface.

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Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body–specific localization signal

The Journal of Cell Biology ◽

10.1083/jcb.200310138 ◽

2004 ◽

Vol 164 (5) ◽

pp. 647-652 ◽

Cited By ~ 167

Author(s):

Beáta E. Jády ◽

Edouard Bertrand ◽

Tamás Kiss

Keyword(s):

S Phase ◽

Cajal Body ◽

Sequence Motif ◽

Telomerase Rna ◽

Dna Repeats ◽

Telomeric Dna ◽

Specific Localization ◽

Localization Signal ◽

Human Telomerase

Telomerase is a ribonucleoprotein reverse transcriptase that uses its RNA component as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes. Here, fluorescence in situ hybridization demonstrates that in HeLa cancer cells, human telomerase RNA (hTR) accumulates in the nucleoplasmic Cajal bodies (CBs). Localization of transiently expressed hTR to CBs is supported by a short sequence motif (411-UGAG-414) that is located in the 3′-terminal box H/ACA RNA-like domain of hTR and that is structurally and functionally indistinguishable from the CB-specific localization signal of box H/ACA small CB-specific RNAs. In synchronized HeLa cells, hTR shows the most efficient accumulation in CBs during S phase, when telomeres are most likely synthesized. CBs may function in post-transcriptional maturation (e.g., cap hypermethylation of hTR), but they may also play a role in the assembly and/or function of telomerase holoenzyme.

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Characterization of Nonspecific Protein−DNA Interactions by1H Paramagnetic Relaxation Enhancement

Journal of the American Chemical Society ◽

10.1021/ja046246b ◽

2004 ◽

Vol 126 (40) ◽

pp. 12800-12808 ◽

Cited By ~ 62

Author(s):

Junji Iwahara ◽

Charles D. Schwieters ◽

G. Marius Clore

Keyword(s):

Paramagnetic Relaxation ◽

Paramagnetic Relaxation Enhancement ◽

Dna Interactions ◽

Protein Dna Interactions

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Characterization of Protein-DNA Interactions Using Protein Microarrays

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot5614 ◽

2011 ◽

Vol 2011 (5) ◽

pp. pdb.prot5614-pdb.prot5614 ◽

Cited By ~ 10

Author(s):

S. Hu ◽

Z. Xie ◽

S. Blackshaw ◽

J. Qian ◽

H. Zhu

Keyword(s):

Protein Microarrays ◽

Dna Interactions ◽

Protein Dna Interactions

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Characterization of Interactions between PinX1 and Human Telomerase Subunits hTERT and hTR

Journal of Biological Chemistry ◽

10.1074/jbc.m408131200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 51745-51748 ◽

Cited By ~ 61

Author(s):

Soma S. R. Banik ◽

Christopher M. Counter

Keyword(s):

Telomerase Activity ◽

Protein Subunit ◽

Human Telomerase Reverse Transcriptase ◽

Telomere Elongation ◽

Cellular Context ◽

Core Components ◽

Human Telomerase

The addition of telomeric repeats to chromosome ends by the enzyme telomerase is a highly orchestrated process. Although much is known regarding telomerase catalytic activityin vitro, less is known about how this activity is regulatedin vivoto ensure proper telomere elongation. One protein that appears to be involved in negatively regulating telomerase functionin vivois PinX1 because overexpression of PinX1 inhibits telomerase activity and causes telomere shortening. To understand the nature of this repression, we characterized the interactions among PinX1 and the core components of telomerase, the human telomerase reverse transcriptase (hTERT) and associated human telomerase RNA (hTR). We now show thatin vitroPinX1 binds directly to the hTERT protein subunit, primarily to the hTR-binding domain, as well as to the hTR subunit. However, in a cellular context, the association of PinX1 with hTR is dependent on the presence of hTERT. Taken together, we suggest that PinX1 represses telomerase activityin vivoby binding to the assembled hTERT·hTR complex.

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