title: Shared Subunits of Tetrahymena Telomerase Holoenzyme and Replication Protein A Have Different Functions in Different Cellular Complexes.
abstract: In most eukaryotes, telomere maintenance relies on telomeric repeat synthesis by a reverse transcriptase named telomerase. To synthesize telomeric repeats, the catalytic subunit telomerase reverse transcriptase (TERT) uses the RNA subunit (TER) as a template. In the ciliate Tetrahymena thermophila, the telomerase holoenzyme consists of TER, TERT, and eight additional proteins, including the telomeric repeat single-stranded DNA-binding protein Teb1 and its heterotrimer partners Teb2 and Teb3. Teb1 is paralogous to the large subunit of the general single-stranded DNA binding heterotrimer replication protein A (RPA). Little is known about the function of Teb2 and Teb3, which are structurally homologous to the RPA middle and small subunits, respectively. Here, epitope-tagging Teb2 and Teb3 expressed at their endogenous gene loci enabled affinity purifications that revealed that, unlike other Tetrahymena telomerase holoenzyme subunits, Teb2 and Teb3 are not telomerase-specific. Teb2 and Teb3 assembled into other heterotrimer complexes, which when recombinantly expressed had the general single-stranded DNA binding activity of RPA complexes, unlike the telomere-specific DNA binding of Teb1 or the TEB heterotrimer of Teb1, Teb2, and Teb3. TEB had no more DNA binding affinity than Teb1 alone. In contrast, heterotrimers reconstituted with Teb2 and Teb3 and two other Tetrahymena RPA large subunit paralogs had higher DNA binding affinity than their large subunit alone. Teb1 and TEB, but not RPA, increased telomerase processivity. We conclude that in the telomerase holoenzyme, instead of binding DNA, Teb2 and Teb3 are Teb1 assembly factors. These findings demonstrate that Tetrahymena telomerase holoenzyme and RPA complexes share subunits and that RPA subunits have distinct functions in different heterotrimer assemblies.
authors.affiliation: From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202 and.
authors.forename: Heather E
authors.initials: HE
authors.lastname: Upton
authors.affiliation: the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095.
authors.forename: Henry
authors.initials: H
authors.lastname: Chan
authors.affiliation: the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095.
authors.forename: Juli
authors.initials: J
authors.lastname: Feigon
authors.affiliation: From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202 and kcollins@berkeley.edu.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Collins
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019098:Telomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics*; D034501:Telomere-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000201:enzymology*
chemical_list: D004277:DNA, Single-Stranded; D020035:Holoenzymes; D021122:Protein Subunits; D051716:Replication Protein A; D034501:Telomere-Binding Proteins; D012313:RNA; D019098:Telomerase
keywords: DNA; chromosomes; protozoan; telomerase; telomere