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Multiple DNA-binding sites in Tetrahymena telomerase.

Finger SN, Bryan TM - Nucleic Acids Res. (2008)

Bottom Line: Both the K(m) and K(d) increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites.However, there appears to be cooperativity between the TEN and RNA-binding domains.Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle.

View Article: PubMed Central - PubMed

Affiliation: Children's Medical Research Institute, 214 Hawkesbury Road, Westmead NSW 2145, Australia.

ABSTRACT
Telomerase is a ribonucleoprotein enzyme that maintains chromosome ends through de novo addition of telomeric DNA. The ability of telomerase to interact with its DNA substrate at sites outside its catalytic centre ('anchor sites') is important for its unique ability to undergo repeat addition processivity. We have developed a direct and quantitative equilibrium primer-binding assay to measure DNA-binding affinities of regions of the catalytic protein subunit of recombinant Tetrahymena telomerase (TERT). There are specific telomeric DNA-binding sites in at least four regions of TERT (the TEN, RBD, RT and C-terminal domains). Together, these sites contribute to specific and high-affinity DNA binding, with a K(d) of approximately 8 nM. Both the K(m) and K(d) increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites. The N-terminal TEN domain, which has previously been implicated in DNA binding, shows only low affinity binding. However, there appears to be cooperativity between the TEN and RNA-binding domains. Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle.

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Related in: MedlinePlus

DNA interacts with telomerase independently of telomerase RNA. (A) The indicated concentrations of biotinylated 18GTT oligonucleotide were incubated with 35S-labelled full-length recombinant TERT (-telomerase RNA) and recovered on NeutrAvidin beads. ‘Input’ represents 20% of the starting material in the bound lanes. LC: 33P-labelled PBR-48-Bio oligonucleotide, used as a loading and recovery control. (B) Quantification of data in A, together with that in Figure 2A, to show increase in Kd in the absence of telomerase RNA. Filled square, binding of Bio-18GTT to TERT + RNA, filled circle, binding of Bio-18GTT to TERT–RNA. The mean of three to four independent experiments is plotted; error bars = SD.
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Figure 3: DNA interacts with telomerase independently of telomerase RNA. (A) The indicated concentrations of biotinylated 18GTT oligonucleotide were incubated with 35S-labelled full-length recombinant TERT (-telomerase RNA) and recovered on NeutrAvidin beads. ‘Input’ represents 20% of the starting material in the bound lanes. LC: 33P-labelled PBR-48-Bio oligonucleotide, used as a loading and recovery control. (B) Quantification of data in A, together with that in Figure 2A, to show increase in Kd in the absence of telomerase RNA. Filled square, binding of Bio-18GTT to TERT + RNA, filled circle, binding of Bio-18GTT to TERT–RNA. The mean of three to four independent experiments is plotted; error bars = SD.

Mentions: It has been reported that yeast and Tetrahymena telomerase are dependent on the telomerase RNA subunit for crosslinking to a DNA primer (22,24). We found that binding of TERT to 18GTT DNA in our binding assay was only partially dependent on the RNA (Figure 3). The Kd for this interaction was 29 ± 5 nM, 3–4-fold greater than that in the presence of RNA. Under these reconstitution conditions, about 50% of TERT protein in the +RNA samples is not bound to telomerase RNA (37), so the actual difference in binding affinities may be somewhat greater than 3–4-fold. Nevertheless, these data clearly show that telomerase RNA contributes to, but is not completely necessary for, binding of Tetrahymena telomerase to a DNA primer.Figure 3.


Multiple DNA-binding sites in Tetrahymena telomerase.

Finger SN, Bryan TM - Nucleic Acids Res. (2008)

DNA interacts with telomerase independently of telomerase RNA. (A) The indicated concentrations of biotinylated 18GTT oligonucleotide were incubated with 35S-labelled full-length recombinant TERT (-telomerase RNA) and recovered on NeutrAvidin beads. ‘Input’ represents 20% of the starting material in the bound lanes. LC: 33P-labelled PBR-48-Bio oligonucleotide, used as a loading and recovery control. (B) Quantification of data in A, together with that in Figure 2A, to show increase in Kd in the absence of telomerase RNA. Filled square, binding of Bio-18GTT to TERT + RNA, filled circle, binding of Bio-18GTT to TERT–RNA. The mean of three to four independent experiments is plotted; error bars = SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275084&req=5

Figure 3: DNA interacts with telomerase independently of telomerase RNA. (A) The indicated concentrations of biotinylated 18GTT oligonucleotide were incubated with 35S-labelled full-length recombinant TERT (-telomerase RNA) and recovered on NeutrAvidin beads. ‘Input’ represents 20% of the starting material in the bound lanes. LC: 33P-labelled PBR-48-Bio oligonucleotide, used as a loading and recovery control. (B) Quantification of data in A, together with that in Figure 2A, to show increase in Kd in the absence of telomerase RNA. Filled square, binding of Bio-18GTT to TERT + RNA, filled circle, binding of Bio-18GTT to TERT–RNA. The mean of three to four independent experiments is plotted; error bars = SD.
Mentions: It has been reported that yeast and Tetrahymena telomerase are dependent on the telomerase RNA subunit for crosslinking to a DNA primer (22,24). We found that binding of TERT to 18GTT DNA in our binding assay was only partially dependent on the RNA (Figure 3). The Kd for this interaction was 29 ± 5 nM, 3–4-fold greater than that in the presence of RNA. Under these reconstitution conditions, about 50% of TERT protein in the +RNA samples is not bound to telomerase RNA (37), so the actual difference in binding affinities may be somewhat greater than 3–4-fold. Nevertheless, these data clearly show that telomerase RNA contributes to, but is not completely necessary for, binding of Tetrahymena telomerase to a DNA primer.Figure 3.

Bottom Line: Both the K(m) and K(d) increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites.However, there appears to be cooperativity between the TEN and RNA-binding domains.Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle.

View Article: PubMed Central - PubMed

Affiliation: Children's Medical Research Institute, 214 Hawkesbury Road, Westmead NSW 2145, Australia.

ABSTRACT
Telomerase is a ribonucleoprotein enzyme that maintains chromosome ends through de novo addition of telomeric DNA. The ability of telomerase to interact with its DNA substrate at sites outside its catalytic centre ('anchor sites') is important for its unique ability to undergo repeat addition processivity. We have developed a direct and quantitative equilibrium primer-binding assay to measure DNA-binding affinities of regions of the catalytic protein subunit of recombinant Tetrahymena telomerase (TERT). There are specific telomeric DNA-binding sites in at least four regions of TERT (the TEN, RBD, RT and C-terminal domains). Together, these sites contribute to specific and high-affinity DNA binding, with a K(d) of approximately 8 nM. Both the K(m) and K(d) increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites. The N-terminal TEN domain, which has previously been implicated in DNA binding, shows only low affinity binding. However, there appears to be cooperativity between the TEN and RNA-binding domains. Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle.

Show MeSH
Related in: MedlinePlus