DNA Binding Affinity and Sequence Permutation Preference of the Telomere Protein from Euplotes crassus

Telomere end binding proteins from diverse organisms use various forms of an ancient protein structure to recognize and bind with single-strand DNA found at the ends of telomeres. To further understand the biochemistry and evolution of these proteins, we have characterized the DNA binding properties...

Full description

Saved in:
Bibliographic Details
Published in:Biochemistry (Easton) 2006-07, Vol.45 (28), p.8628-8638
Main Authors: Suzuki, Takahito, McKenzie, Margaret, Ott, Elizabeth, Ilkun, Olesya, Horvath, Martin P
Format: Article
Language:English
Subjects:
Animals
Cloning, Molecular
DNA, Single-Stranded - chemistry
DNA, Single-Stranded - metabolism
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Escherichia coli
Escherichia coli - genetics
Euplotes
Euplotes - metabolism
Euplotes crassus
Oxytricha nova
Phylogeny
Protein Structure, Tertiary
Protozoan Proteins - chemistry
Protozoan Proteins - genetics
Protozoan Proteins - metabolism
Telomere - chemistry
Telomere - genetics
Telomere - metabolism
Telomere-Binding Proteins - chemistry
Telomere-Binding Proteins - genetics
Telomere-Binding Proteins - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Telomere end binding proteins from diverse organisms use various forms of an ancient protein structure to recognize and bind with single-strand DNA found at the ends of telomeres. To further understand the biochemistry and evolution of these proteins, we have characterized the DNA binding properties of the telomere end binding protein from Euplotes crassus (EcTEBP). EcTEBP and its predicted amino-terminal DNA-binding domain, EcTEBP-N, were expressed in Escherichia coli and purified. Each protein formed stoichiometric (1:1) complexes with single-strand DNA oligos derived from the precisely defined d(TTTTGGGGTTTTGG) sequence found at DNA termini in Euplotes. Dissociation constants for DNA·EcTEBP and DNA·EcTEBP-N complexes were comparable:  K D-DNA = 38 ± 2 nM for the full-length protein and K D - DNA = 60 ± 4 nM for the N-terminal domain, indicating that the N-terminal domain retains a high affinity for DNA even in the absence of potentially stabilizing moieties located in the C-terminal domain. Rate constants for DNA association and DNA dissociation corroborated a slightly improved DNA binding performance for the full-length protein (k a = 45 ± 4 μM-1 s-1, k d = 0.10 ± 0.02 s-1) relative to that of the N-terminal domain (k a = 18 ± 1 μM-1 s-1, k d = 0.15 ± 0.01 s-1). Equilibrium dissociation constants measured for sequence permutations of the telomere repeat spanned the range of 55−1400 nM, with EcTEBP and EcTEBP-N binding most tightly to d(TTGGGGTTTTGG), the sequence corresponding to that of mature DNA termini. Additionally, competition experiments showed that EcTEBP recognizes and binds the telomere-derived 14-nucleotide DNA in preference to shorter 5‘-truncation variants. Compared with the results for multisubunit complexes assembled with telomere single-strand DNA from Oxytricha nova, our results highlight the relative simplicity of the E. crassus system where a telomere end binding protein has biochemical properties indicating one protein subunit caps the single-strand DNA.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi060388w