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PMID: 12364610 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Delineation of the high-affinity single-stranded telomeric DNA-binding domain of Saccharomyces cerevisiae Cdc13.

Nucleic acids research ·Vol. 30 ·No. 19 ·2002-10-01 ·Pages 4305-13

Anderson EM, Halsey WA, Wuttke DS

Abstract

Cdc13 is an essential protein from Saccharomyces cerevisiae that caps telomeres by protecting the C-rich telomeric DNA strand from degradation and facilitates telomeric DNA replication by telomerase. In vitro, Cdc13 binds TG-rich single-stranded telomeric DNA with high affinity and specificity. A previously identified domain of Cdc13 encompassing amino acids 451-694 (the 451-694 DBD) retains the single-stranded DNA-binding properties of the full-length protein; however, this domain contains a large unfolded region identified in heteronuclear NMR experiments. Trypsin digestion and MALDI mass spectrometry were used to identify the minimal DNA-binding domain (the 497-694 DBD) necessary and sufficient for full DNA-binding activity. This domain was completely folded, and the N-terminal unfolded region removed was shown to be dispensable for function. Using affinity photocrosslinking to site-specifically modified telomeric single-stranded DNA, the 497-694 DBD was shown to contact the entire 11mer required for high-affinity binding. Intriguingly, both domains bound single-stranded telomeric DNA with much greater affinity than the full-length protein. The full-length protein exhibited the same rate of dissociation as both domains, however, indicating that the full-length protein contains a region that inhibits association with single-stranded telomeric DNA.

MeSH Terms
Binding Sites/genetics Binding, Competitive DNA, Recombinant/genetics,metabolism DNA, Single-Stranded/metabolism Electrophoresis, Polyacrylamide Gel Electrophoretic Mobility Shift Assay Oligonucleotides/genetics,metabolism Saccharomyces cerevisiae Proteins/genetics,metabolism Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Telomere/genetics,metabolism Telomere-Binding Proteins/genetics,metabolism
Chemicals
Cdc13 protein, S cerevisiae DNA, Recombinant DNA, Single-Stranded Oligonucleotides Saccharomyces cerevisiae Proteins Telomere-Binding Proteins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Anderson Emily M
Department of Chemistry and Biochemistry, University of Colorado at Boulder, UCB 215, Boulder, CO 80309-0215, USA.
Halsey Wayne A
Wuttke Deborah S
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkf554
Published
2002-10-01
Pages
4305-13
Language
English
Region
England
NLM ID
0411011
PMCID
PMC140553
Subset
IM
Grants
NIGMS NIH HHS · R01 GM059414 · United States
NCRR NIH HHS · S10 RR011969 · United States
NIGMS NIH HHS · GM59414 · United States
External Links
PubMed source DOI: 10.1093/nar/gkf554 PMC Full Text View journal details
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