Structural basis for Tetrahymena telomerase processivity factor Teb1 binding to single-stranded telomeric-repeat DNA

Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20357-61. doi: 10.1073/pnas.1113624108. Epub 2011 Dec 5.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / genetics*
  • Crystallography, X-Ray / methods
  • DNA / genetics*
  • DNA, Single-Stranded / genetics*
  • DNA-Binding Proteins / genetics*
  • Humans
  • Models, Genetic
  • Models, Molecular
  • Molecular Conformation
  • Replication Protein A / chemistry
  • Telomerase / genetics*
  • Telomere / ultrastructure*
  • Telomere-Binding Proteins / chemistry
  • Tetrahymena / enzymology*

Substances

  • Bacterial Proteins
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • RPA1 protein, human
  • Replication Protein A
  • Telomere-Binding Proteins
  • DNA
  • Telomerase

Associated data

  • PDB/3U4V
  • PDB/3U4Z
  • PDB/3U50
  • PDB/3U58