Delineation of the high-affinity single-stranded telomeric DNA-binding domain of Saccharomyces cerevisiae Cdc13.
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.
D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016615:Telomere / Q000235:genetics / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism*
C452219:Cdc13 protein, S cerevisiae; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D034501:Telomere-Binding Proteins