Nucleic Acids Research, 1989, Vol. 17, No. 11 4235-4253
© 1989
MOLECULAR BIOLOGY |
Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA
1Department of Molecular, Cellular and Developmental Biology Boulder, CO 80309-0215, USA Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado Boulder, CO 80309-0215, USA
Received March 1, 1989. Revised May 9, 1989. Accepted May 9, 1989.
The telomere binding protein from macronuclei of Oxytricha nova binds macronuclear DNA in vitro, protecting the 3'-terminal single-stranded (T4G4)2 tail from chemical and enzymatic probes. We have used synthetic oligodeoxynucleotides to study the binding properties of the telomere protein. It binds at the 3' end of single-stranded oligonucleotides that have the sequence (T4G4)n, where n
2, reconstituting the methylation protection seen with macronuclear DNA. Three oligonucleotide.protein complexes are resolved in nondenaturing gels, all specific for this sequence. Single-stranded oligonucleotides that have one or more repeats of the sequence C4A4 are also recognized, forming a single complex. The dissociation constant for (T4G4)4 is about 19 nM, and for macronuclear DNA is at least 20-fold lower. The basis for this difference is not fully understood, but it is not simply due to the absence of a (C4A4)2.5.(G4T4)2.5 region on the oligonucleotide. Transversions of T's to A's or of G's to C's in the 3' tail portion prevent binding. Changing T's to dU's does not prevent binding, indicating that the hydrophobic 5-methyl group is not required for binding as had been suggested from the salt-stability of the complex. The properties of the DNA-protein complex suggest a revised model for telomere synthesis in Oxytricha.
*Present address: Department of Chemistry, Northern Arizona University, Flagstaff, AZ 86011, USA
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