title: Structural basis for Tetrahymena telomerase processivity factor Teb1 binding to single-stranded telomeric-repeat DNA.
abstract: Telomerase copies its internal RNA template to synthesize telomeric DNA repeats. Unlike other polymerases, telomerase can retain its single-stranded product through multiple rounds of template dissociation and repositioning to accomplish repeat addition processivity (RAP). Tetrahymena telomerase holoenzyme RAP depends on a subunit, Teb1, with autonomous DNA-binding activity. Sequence homology and domain modeling suggest that Teb1 is a paralog of RPA70C, the largest subunit of the single-stranded DNA-binding factor replication protein (RPA), but unlike RPA, Teb1 binds DNA with high specificity for telomeric repeats. To understand the structural basis and significance of telomeric-repeat DNA recognition by Teb1, we solved crystal structures of three proposed Teb1 DNA-binding domains and defined amino acids of each domain that contribute to DNA interaction. Our studies indicate that two central Teb1 DNA-binding oligonucleotide/oligosaccharide-binding-fold domains, Teb1A and Teb1B, achieve high affinity and selectivity of telomeric-repeat recognition by principles similar to the telomere end-capping protein POT1 (protection of telomeres 1). An additional C-terminal Teb1 oligonucleotide/oligosaccharide-binding-fold domain, Teb1C, has features shared with the RPA70 C-terminal domain including a putative direct DNA-binding surface that is critical for high-RAP activity of reconstituted holoenzyme. The Teb1C zinc ribbon motif does not contribute to DNA binding but is nonetheless required for high-RAP activity, perhaps contributing to Teb1 physical association with the remainder of the holoenzyme. Our results suggest the biological model that high-affinity DNA binding by Teb1AB recruits holoenzyme to telomeres and subsequent Teb1C-DNA association traps product in a sliding-clamp-like manner that does not require high-affinity DNA binding for high stability of enzyme-product association.
authors.affiliation: Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Zhixiong
authors.initials: Z
authors.lastname: Zeng
authors.forename: Bosun
authors.initials: B
authors.lastname: Min
authors.forename: Jing
authors.initials: J
authors.lastname: Huang
authors.forename: Kyungah
authors.initials: K
authors.lastname: Hong
authors.forename: Yuting
authors.initials: Y
authors.lastname: Yang
authors.forename: Kathleen
authors.initials: K
authors.lastname: Collins
authors.forename: Ming
authors.initials: M
authors.lastname: Lei
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D018360:Crystallography, X-Ray / Q000379:methods; D004247:DNA / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D008958:Models, Molecular; D008968:Molecular Conformation; D051716:Replication Protein A / Q000737:chemistry; D019098:Telomerase / Q000235:genetics*; D016615:Telomere / Q000648:ultrastructure*; D034501:Telomere-Binding Proteins / Q000737:chemistry; D013768:Tetrahymena / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D034501:Telomere-Binding Proteins; D004247:DNA; D019098:Telomerase