DNA recognition and binding by the Euplotes telomere protein

Abstract

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.

DNA Binding Affinity and Sequence Permutation Preference of the Telomere Protein from Euplotes crassus

“…The DNA-binding properties of recombinant EcTEBP and EcTEBP-N were, to some degree, similar to those observed previously for protein isolated directly from Euplotes crassus (34). Because of improved yield and solubility of the bacterially expressed proteins, we could for the first time determine binding stoichiometries, equilibrium dissociation constants and kinetic rate constants and further evaluate how DNA length and sequence register relate to DNAbinding affinity.…”

“…In our studies, we either used short oligos with at most one full d(GGGG) tract or replaced sodium ions with lithium ions, and in this way we could perform binding experiments at close to physiological ionic strength without complications arising from structured DNA. Despite these experimental differences, both the current and previous work reach the same important conclusion that the telomere end binding protein has biochemical properties well suited to recognize, bind and protect the single strand DNA termini of telomeres in Euplotes crassus (34). The advantages of our recombinant expression system allowed for quantitative measures of DNA binding not previously possible, which is significant because we can now make new comparisons with the telomere binding systems of other organisms.…”

“…EcTEBP purified from its natural source bound T and G-rich single strand DNA, but binding of the 14-nucleotide d(TTTTGGGGTTTTGG) DNA required attachment of this site to a longer piece of either single-stranded or double-stranded DNA (34). Additionally, while the fulllength protein isolated from Euplotes crassus showed differential binding of telomere repeat DNAs ending with d(G 2 ) and d(T 4 ), the N-terminal domain prepared by trypsin digestion appeared to have diminished sequence specificity and formed complexes with both DNA sequences (34). In our experiments, short oligos formed DNA-protein complexes as determined by several methods, and the N-terminal domain, EcTEBP-N, recapitulated DNA-binding properties measured for the full-length protein, including telomere sequence permutation preference (see Table III).…”

“…Protein purified from Euplotes crassus was susceptible to aggregation especially as sensitized by detergents used in extraction steps (34). Solubility of recombinantly expressed protein was also a concern, overcome ultimately by keeping the preparation cold and avoiding excursions to room temperature during induction, extraction and purification steps.…”

“…This DNA can also bind one or two alpha subunits in the absence of beta (24,31,32). In Euplotes crassus, the single strand DNA forms a complex with a telomere end binding protein (33,34) and may also interact with a second telomere protein homologue during DNA synthesis (35,36). Figure 1 shows a phylogenetic relationship deduced from amino acid sequences of the ciliate telomere end binding proteins and telomere binding protein homologues.…”