Properties of the telomeric DNA-binding protein from Oxytricha nova.
Telomeres of Oxytricha macronuclear DNA exist as discrete DNA-protein complexes. Different regions of each complex display characteristic DNA-protein interactions. In the most terminal region, binding of a 43- and a 55-kDa protein to the telomeric DNA appears to account for all the DNA-protein interactions that can be detected by chemical and nuclease footprinting. We have used gradient sedimentation and protein-protein cross-linking to establish that the 43- and 55-kDa proteins are subunits of a heterodimer. Both subunits are very basic, which is unexpected considering the resistance of the DNA-protein interaction to high concentrations of salt. It is extremely difficult to dissociate the two subunits either from telomeric DNA or from each other. Even after extensive treatment of protein preparations with nuclease, a fragment of the 3' tail from macronuclear DNA remains bound to the protein. A wide range of conditions was screened for dissociation of the subunits from the DNA and/or from each other. Dissociation was only obtained by using conditions that caused some inactivation of the DNA-binding capacity of the protein. The use of reagents that covalently modify sulfydryl groups during the purification procedure facilitates preparation of telomere protein with full DNA-binding activity.
Price
C M
CM
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Cech
T R
TR
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000648:ultrastructure; D002875:Chromosomes / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D056890:Eukaryota / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D013388:Succinimides / Q000378:metabolism
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D013388:Succinimides; C011240:dithiobis(succinimidylpropionate)