Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain

Buczek, Paweł; Horvath, Martin P.

doi:10.1016/j.jmb.2006.02.082

Cited by 16 publications

(25 citation statements)

References 120 publications

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“…2A) was characterized by favorable enthalpy and entropy terms (⌬H 1.1 Ͻ 0, ⌬S 1.1 Ͼ 0) at each temperature tested, making the resulting ␣⅐DNA complex very stable with ⌬G 1.1 ϭ Ϫ9.89 Ϯ 0.03 kcal/ mol at 25°C. Highly similar binding behavior with nearly identical thermodynamic parameters was observed for the N-terminal DNA-binding domain of ␣ binding with the same 11-nt DNA (21), confirming that the N-terminal domain of ␣ is sufficient for DNA binding and indicating that the C-terminal protein-protein association domain of ␣ does not participate significantly in this initial encounter with DNA.…”

Section: Itc For Firstmentioning

confidence: 54%

“…Dissociation of ␤ provides a route to an elongation complex with telomerase; however, this event must be carefully coordinated to avoid ␣ 2 ⅐DNA complexes expected at equilibrium (21) that are also not telomerase substrates (v in Fig. 7) (44).…”

Section: Discussionmentioning

confidence: 99%

“…Analysis by SDS-PAGE confirmed that when handled with these precautions, ␤ remains Ͼ95% intact for at least 4 weeks. DNA oligonucleotides were purified by reverse phase high pressure liquid chromatography in both trityl-protected and deprotected forms as previously described (21). The ␣⅐DNA complex was prepared by mixing molar equivalents of ␣ and DNA, followed by size exclusion chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…Previous ITC studies of single-stranded telomere DNA demonstrated that high quality data could be obtained most consistently over a broad range of temperatures if the DNA component was included within the sample cell (21), and this constraint directed our choice of titrant and sample cell components. Typically, DNA or DNA-protein complex was diluted to 30 M, degassed, and placed in the sample cell.…”

Section: Methodsmentioning

confidence: 99%

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Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova

Buczek

Horvath

2006

Journal of Biological Chemistry

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In Sterkiella nova, ␣ and ␤ telomere proteins bind cooperatively with single-stranded DNA to form a ternary ␣⅐␤⅐DNA complex. Association of telomere protein subunits is DNAdependent, and ␣-␤ association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, ␣ and DNA first form a stable ␣⅐DNA complex followed by the addition of ␤ in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with ⌬Cp ‫؍‬ ؊305 ؎ 3 cal mol ؊1 K ؊1 for ␣ binding with DNA and ⌬Cp ‫؍‬ ؊2010 ؎ 20 cal mol ؊1 K ؊1 for the addition of ␤ to complete the ␣⅐␤⅐DNA complex. By examining alternate routes including titration of singlestranded DNA with a preformed ␣⅐␤ complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of ␤. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length ␤ or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of ␤.Telomere nucleoprotein complexes serve critical functions in eukaryotic cells, protecting the ends of chromosomes from degradation, recombination, and end-to-end fusion events (1-3). A conserved feature of telomere DNA is a singlestranded 3Ј-terminal extension (4 -7) that acts as the primer for synthesis of telomere DNA repeats catalyzed by the ribonucleoprotein enzyme telomerase (8). By compensating for DNA loss accompanying lagging strand DNA synthesis, telomerase-mediated extension of telomere DNA ensures that genetic information in chromosomes is completely replicated with each cell division.Telomeres found in the macronuclei of Sterkiella nova (Oxytricha nova formerly) are relatively simple and well defined yet possess higher levels of structural organization and therefore represent an important model system for understanding the structures of telomere ends. Telomere DNA in S. nova consists of 20 base pairs of double-stranded DNA followed by 16 nucleotides of 3Ј-terminal single-stranded DNA of sequence d(TTTTGGGGTTTTGGGG) (4). The single-stranded DNA forms a tenacious salt-resistant complex with two protein subunits, a 56-kDa protein called ␣ and a 41-kDa protein called ␤ (9 -12). The ␣ protein contains two structurally and functionally separable domains, a 35-kDa N-terminal DNA-binding domain and a 21-kDa C-terminal domain necessary for protein-protein association (13). The ...

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Section: Itc For Firstmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova

Buczek

Horvath

2006

Journal of Biological Chemistry

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“…In Oxytricha nova, the single strand DNA forms a ternary complex with one alpha subunit and one beta subunit (20,24,29,30). This DNA can also bind one or two alpha subunits in the absence of beta (24,31,32). In Euplotes crassus, the single strand DNA forms a complex with a telomere end binding protein (33,34) and may also interact with a second telomere protein homologue during DNA synthesis (35,36).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2006

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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 E. 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 were comparable, with 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 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 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 a 55 -1400 nM range, with EcTEBP and EcTEBP-N binding most tightly to d (TTGGGGTTTTGG) -the sequence corresponding with 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 multi-subunit complexes assembled with telomere single strand DNA from Oxytricha nova, our results highlight the relative simplicity of the Euplotes crassus system where a telomere end binding protein has biochemical properties indicating one protein subunit caps the single strand DNA.

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Design and Evolution of Artificial Metalloenzymes: Biomimetic Aspects

Creus¹,

Ward²

2011

Progress in Inorganic Chemistry

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Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain

Cited by 16 publications

References 120 publications

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova

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

Design and Evolution of Artificial Metalloenzymes: Biomimetic Aspects

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