Interference Footprinting Analysis of Telomerase Elongation Complexes

Abstract

Telomerase is a reverse transcriptase that adds single-stranded telomeric repeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing reverse transcriptase motifs, and auxiliary proteins. We have carried out an interference footprinting analysis of the Tetrahymena telomerase elongation complexes. In this study, single-stranded oligonucleotide primers containing telomeric sequences were modified with base-specific chemical reagents and extended with the telomerase by a single 32 P-labeled dGMP or dTMP. Base modifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the telomerase and the six or seven 3-terminal residues of the primers. These interactions occurred not only with the RNA template region, but also with another region in the enzyme ribonucleoprotein complex designated the telomerase DNA interacting surface (TDIS). This was indicated by footprints generated with dimethyl sulfate (that did not affect Watson-Crick hydrogen bonding) and by footprinting assays performed with mutant primers. In primers aligned at a distance of 2 nucleotides along the RNA template region, the footprints of the six or seven 3-terminal residues were shifted by 2 nucleotides. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3 ends of the primers relative to the template region. Weak interactions occurred between the telomerase and residues located upstream of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase

“…2 B-E). This apparent uncoupling of the register of DNA-TERT interaction from the register of DNA-template interaction is consistent with results from our previous DNA footprinting and kinetic assays (10,18,19).…”

“…This position was previously shown to be involved in primer interaction with telomerase (10,13). A complex of reconstituted core enzyme (tTERT and tTER) with primer was irradiated with a long-wavelength UV light (Ͼ295 nm), thereby favoring crosslinks of the IdU with aromatic amino acids in close proximity.…”

“…More direct physical assays have also been used to investigate enzyme-primer interactions. Our previous interference footprinting studies indicated that functionally nonredundant interactions of primer with enzyme occur primarily in the six or seven 3Ј-terminal primer nucleotides (10). In addition, atomic-resolution structure determined for residues 13-176 of the tTERT TEN domain revealed a surface groove with features suggestive of a channel for binding single-stranded DNA (11).…”

Requirements for the dGTP-dependent Repeat Addition Processivity of Recombinant Tetrahymena Telomerase

“…8). The specificity of the nascent product interaction would be reflected in the template and/or product sequence requirements described above and possibly by the inhibition of primer use upon dimethyl sulfate modification of terminal guanosines at the N-7 position, which is not predicted to affect base pairing with the template (25). If nascent product interaction can persist in the absence of a template hybrid, the proposed telomerase nascent product binding site would more closely resemble that of an RNA polymerase than a viral RT, which interacts with product in the context of the minor groove of a template-product hybrid (26).…”

Mapping of DNA Binding Sites in the Tetrahymena Telomerase Holoenzyme Proteins by UV Cross-Linking and Mass Spectrometry

“…Using for these assays oligo primers aligned at different positions along the template, we identified strong interactions of both types of enzyme with six to seven nucleotides at the 3′-termini of the primers. [16][17][18] Subsequently, we used ultraviolet light (UV) cross-linking techniques and site-directed mutagenesis to map amino acids in TERT that interact with DNA primers in complexes generated with the reconstituted core enzyme. These studies revealed a major interaction between the amino acid tryptophan 187 (W187) in the TEN domain of TERT and primer nucleotides aligned at the beginning of the RNA template region.…”