Characterization of Nonspecific Protein−DNA Interactions by1H Paramagnetic Relaxation Enhancement

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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ChemInform Abstract: Theory, Practice, and Applications of Paramagnetic Relaxation Enhancement for the Characterization of Transient Low-Population States of Biological Macromolecules and Their Complexes

ChemInform ◽

10.1002/chin.200951280 ◽

2009 ◽

Vol 40 (51) ◽

pp. no-no

Author(s):

G. Marius Clore ◽

Junji Iwahara

Keyword(s):

Paramagnetic Relaxation ◽

Paramagnetic Relaxation Enhancement ◽

Biological Macromolecules ◽

Abstract Theory

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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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Paramagnetic relaxation enhancement for the characterization of the conformational heterogeneity in two-domain proteins

Physical Chemistry Chemical Physics ◽

10.1039/c2cp40139h ◽

2012 ◽

Vol 14 (25) ◽

pp. 9149 ◽

Cited By ~ 32

Author(s):

Ivano Bertini ◽

Claudio Luchinat ◽

Malini Nagulapalli ◽

Giacomo Parigi ◽

Enrico Ravera

Keyword(s):

Paramagnetic Relaxation ◽

Paramagnetic Relaxation Enhancement ◽

Conformational Heterogeneity

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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 Physical and Functional Anchor Site Interactions in Human Telomerase

Molecular and Cellular Biology ◽

10.1128/mcb.02368-06 ◽

2007 ◽

Vol 27 (8) ◽

pp. 3226-3240 ◽

Cited By ~ 48

Author(s):

Haley D. M. Wyatt ◽

Deirdre A. Lobb ◽

Tara L. Beattie

Keyword(s):

Critical Role ◽

Telomerase Rna ◽

Dna Interactions ◽

Telomeric Dna ◽

Protein Subunit ◽

Protein Dna Interactions ◽

N Terminus ◽

Human Telomerase ◽

Linear Chromosomes

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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