Limits...
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.

Show MeSH

Related in: MedlinePlus

Crosslinking of 5-iodo-deoxyuridine substituted 20GTT to TERT and fragments of TERT. (A) Sequence of the four oligonucleotides used for crosslinking in B–F; IU = 5-iodo-deoxyuridine. (B) 32P-labelled DNA (15 nM) was crosslinked to full-length TERT in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. Crosslinking was carried out in the presence or absence of telomerase RNA. (C) 32P-labelled DNA (90 nM) was crosslinked to N519 in the absence of competitor or in the presence of a 55-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (D) 32P-labelled DNA (15 nM) was crosslinked to C598 in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (E) 32P-labelled DNA (100 nM) was crosslinked to RBD in the absence of competitor or in the presence of a 50-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (F) 32P-labelled DNA (50 nM) was crosslinked to full-length TERT, ΔTEN or a 1:1 mixture of both proteins in the presence of a 100-fold excess of cold non-telomeric (PBR) competitor primer. The upper panel shows the signal from 32P-labelled DNA only (with a piece of X-ray film between the gel and phosphorimager screen to shield 35S signals); the lower panel shows signals from both 32P and 35S-labelled proteins, to ensure the use of an equimolar mixture of proteins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2275084&req=5

Figure 5: Crosslinking of 5-iodo-deoxyuridine substituted 20GTT to TERT and fragments of TERT. (A) Sequence of the four oligonucleotides used for crosslinking in B–F; IU = 5-iodo-deoxyuridine. (B) 32P-labelled DNA (15 nM) was crosslinked to full-length TERT in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. Crosslinking was carried out in the presence or absence of telomerase RNA. (C) 32P-labelled DNA (90 nM) was crosslinked to N519 in the absence of competitor or in the presence of a 55-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (D) 32P-labelled DNA (15 nM) was crosslinked to C598 in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (E) 32P-labelled DNA (100 nM) was crosslinked to RBD in the absence of competitor or in the presence of a 50-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (F) 32P-labelled DNA (50 nM) was crosslinked to full-length TERT, ΔTEN or a 1:1 mixture of both proteins in the presence of a 100-fold excess of cold non-telomeric (PBR) competitor primer. The upper panel shows the signal from 32P-labelled DNA only (with a piece of X-ray film between the gel and phosphorimager screen to shield 35S signals); the lower panel shows signals from both 32P and 35S-labelled proteins, to ensure the use of an equimolar mixture of proteins.

Mentions: All sequences are listed 5′ to 3′. Some oligonucleotides were modified by addition of a biotin, on either the 5′ (e.g. Bio-18GTT) or the 3′ (e.g. PBR48-Bio) end. 20GTT was modified by incorporation of 5-iodo-deoxyuridine (Figure 5).


Multiple DNA-binding sites in Tetrahymena telomerase.

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

Crosslinking of 5-iodo-deoxyuridine substituted 20GTT to TERT and fragments of TERT. (A) Sequence of the four oligonucleotides used for crosslinking in B–F; IU = 5-iodo-deoxyuridine. (B) 32P-labelled DNA (15 nM) was crosslinked to full-length TERT in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. Crosslinking was carried out in the presence or absence of telomerase RNA. (C) 32P-labelled DNA (90 nM) was crosslinked to N519 in the absence of competitor or in the presence of a 55-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (D) 32P-labelled DNA (15 nM) was crosslinked to C598 in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (E) 32P-labelled DNA (100 nM) was crosslinked to RBD in the absence of competitor or in the presence of a 50-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (F) 32P-labelled DNA (50 nM) was crosslinked to full-length TERT, ΔTEN or a 1:1 mixture of both proteins in the presence of a 100-fold excess of cold non-telomeric (PBR) competitor primer. The upper panel shows the signal from 32P-labelled DNA only (with a piece of X-ray film between the gel and phosphorimager screen to shield 35S signals); the lower panel shows signals from both 32P and 35S-labelled proteins, to ensure the use of an equimolar mixture of proteins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Crosslinking of 5-iodo-deoxyuridine substituted 20GTT to TERT and fragments of TERT. (A) Sequence of the four oligonucleotides used for crosslinking in B–F; IU = 5-iodo-deoxyuridine. (B) 32P-labelled DNA (15 nM) was crosslinked to full-length TERT in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. Crosslinking was carried out in the presence or absence of telomerase RNA. (C) 32P-labelled DNA (90 nM) was crosslinked to N519 in the absence of competitor or in the presence of a 55-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (D) 32P-labelled DNA (15 nM) was crosslinked to C598 in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (E) 32P-labelled DNA (100 nM) was crosslinked to RBD in the absence of competitor or in the presence of a 50-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (F) 32P-labelled DNA (50 nM) was crosslinked to full-length TERT, ΔTEN or a 1:1 mixture of both proteins in the presence of a 100-fold excess of cold non-telomeric (PBR) competitor primer. The upper panel shows the signal from 32P-labelled DNA only (with a piece of X-ray film between the gel and phosphorimager screen to shield 35S signals); the lower panel shows signals from both 32P and 35S-labelled proteins, to ensure the use of an equimolar mixture of proteins.
Mentions: All sequences are listed 5′ to 3′. Some oligonucleotides were modified by addition of a biotin, on either the 5′ (e.g. Bio-18GTT) or the 3′ (e.g. PBR48-Bio) end. 20GTT was modified by incorporation of 5-iodo-deoxyuridine (Figure 5).

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