Binding linkage in a telomere DNA-protein complex at the ends of Oxytricha nova chromosomes.
Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein-protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (K(D-DNA)=1.4 nM). Another fusion protein, constructed without the C-terminal protein-protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (K(D-DNA)=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA-protein stability to protein-protein contacts at a remote site may provide a trigger point for DNA-protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase.
Buczek
Pawel
P
Department of Biology, University of Utah, 257 S 1400 E, Salt Lake City, UT 84112-0840, USA.
Orr
Rochelle S
RS
Pyper
Sean R
SR
Shum
Mili
M
Kimmel
Emily
E
Ota
Irene
I
Gerum
Shawn E
SE
Horvath
Martin P
MP
D000818:Animals; D002875:Chromosomes / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D046912:Multiprotein Complexes; D016813:Oxytricha / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D021122:Protein Subunits; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D016615:Telomere / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000378:metabolism*
D046912:Multiprotein Complexes; D021122:Protein Subunits; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D034501:Telomere-Binding Proteins; D004247:DNA