DNA recognition and binding by the Euplotes telomere protein.
The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.
Price
C M
CM
Department of Chemistry, University of Nebraska, Lincoln 68588.
Skopp
R
R
Krueger
J
J
Williams
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016054:DNA, Protozoan / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016812:Euplotes / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016813:Oxytricha / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D010446:Peptide Fragments; D015800:Protozoan Proteins; D014357:Trypsin