Investigating the role of the Est3 protein in yeast telomere replication.
The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Delta strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of the S. castellii telomerase RNA. Furthermore, using nuclear magnetic resonance chemical shift perturbation, no chemical shift change was observed at any site in the protein upon addition of single-stranded DNA, arguing against a role for Est3 in recognition of telomeric substrates by telomerase. Addition of exogenous Est3 protein, including mutant Est3 proteins that are severely impaired for telomere replication in vivo, fully restored activity in est3-Delta telomerase reactions. Thus, Est3 performs an in vivo regulatory function in telomere replication, which is distinct from any potential contribution that Est3 might make to telomerase activity.
Lee
Jaesung
J
The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Mandell
Edward K
EK
Rao
Timsi
T
Wuttke
Deborah S
DS
Lundblad
Victoria
V
D004277:DNA, Single-Stranded / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017353:Gene Deletion; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012440:Saccharomyces / Q000201:enzymology* / Q000235:genetics; D019098:Telomerase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016615:Telomere / Q000378:metabolism*
D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D021122:Protein Subunits; D019098:Telomerase