Specific binding of single-stranded telomeric DNA by Cdc13p of Saccharomyces cerevisiae.
Cdc13p is a single strand telomere-binding protein of Saccharomyces cerevisiae; its telomere-binding region is within amino acids 451-693, Cdc13(451-693)p. In this study, we used purified Cdc13p and Cdc13(451-693)p to characterize their telomere binding activity. We found that the binding specificity of single-stranded TG(1-3) DNA by these two proteins is similar. However, the affinity of Cdc13(451-693)p to DNA was slightly lower than that of Cdc13p. The binding of telomeric DNA by these two proteins was disrupted at NaCl concentrations higher than 0.3 m, indicating that electrostatic interaction contributed significantly to the binding process. Because both proteins bound to strand TG(1-3) DNA positioned at the 3' end, the 5' end, or in the middle of the oligonucleotide substrates, our results indicated that the location of TG(1-3) in single-stranded DNA does not appear to be important for Cdc13p binding. Moreover, using DNase I footprint analysis, the structure of the telomeric DNA complexes of Cdc13p and Cdc13(451-693)p was analyzed. The DNase I footprints of these two proteins to three different telomeric DNA substrates were virtually identical, indicating that the telomere contact region of Cdc13p is within Cdc13(451-693)p. Together, the binding properties of Cdc13p and its binding domain support the theory that the specific binding of Cdc13p to telomeres is an important feature of telomeres that regulate telomerase access and/or differentiate natural telomeres from broken ends.
Lin
Y C
YC
Institute of Biopharmaceutical Science, National Yang-Ming University, Shih-Pai, 112 Taipei, Taiwan, Republic of China.
Hsu
C L
CL
Shih
J W
JW
Lin
J J
JJ
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D019926:Cyclin B / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004926:Escherichia coli; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D018411:Spodoptera; D016615:Telomere / Q000378:metabolism*
D019926:Cyclin B; D011994:Recombinant Proteins; D004247:DNA