Nucleic Acids Research, Vol. 20, No. 15 4103-4104

. 1992 Oxford University Press

Rapid isolation of specific DNA-binding proteins DNA-binding domains

and

their

Urszula Wichser and Christine Brack Department of Cell Biology, Biozentrum University Basel, CH-4056 Basel, Switzerland Submitted April 2, 1992

UV-crosslinking of nuclear proteins to labelled oligonucleotides and their separation by SDS-polyacrylamide gel electrophoresis (SDS -PAGE) allows the detection of sequence-specific DNAbinding proteins in nuclear extracts and to determine their molecular size (1, 2). We have developed a simple purification procedure to isolate crosslinked specific protein-DNA complexes from nuclear extracts in sufficient purity to determine the amino terminal sequence of the DNA-bound protein. Oligonucleotides containing the specific binding site are synthesized with a tail of 10 dA at their 3' end. The second strand is synthesized in the presence of 32P-dATP and BrdUTP as described (2). Nuclear extract is incubated with the labelled oligonucleotides under stringent conditions (for details see Figure). After UV crosslinking the complexes are purified over a dT-cellulose column as follows. Oligo dT-cellulose (Pharmacia Type 7, pretreated with 0.1% ficoll, 0.1 % polyvinylpyrrolidon in binding buffer) is suspended in binding buffer, mixed with the UV-crosslinked sample, and incubated on ice for 30 minutes (with occasional gentle shaking) to allow for annealing of the dA tail of the protein-DNA complex to the dT-cellulose, and then loaded onto a column. After washing the column with binding buffer containing 0.4 M NaCl, the complexes are eluted with a low stringency buffer (10 mM Tris, 1 mM EDTA) or with distilled H20. In the example presented in Figure 1 nuclear extract from mouse B-cell hybridoma cells was crosslinked to an immunoglobulin gene promoter oligonucleotide containing the octamer sequence (2). Aliquots of the column fractions were analyzed by SDS -PAGE. Autoradiography of the gel (Figure IA) shows that the eluted fractions contain several protein-DNA complexes. Silver staining of the same gel revealed that most proteins remain in the flow through and wash fractions, and that the eluted fractions do not contain any detectable amounts of protein (Figure 1B). An excess of cold specific promoter oligonucleotide in the reaction prevents formation of the 100 kDa and 60 kDa complexes, nonspecific competitor oligonucleotide does not (Figure IC). In order to analyze individual protein complexes further, fractions are pooled, concentrated by TCA precipitation and loaded onto an SDS gel. After short exposure of the wet gel, individual complexes are blotted to glass fiber filters and used for protein microsequencing (3, 4). We have applied this protocol to isolate directly the DNA binding domain of a mouse octamer binding protein (mOct-2). Previous experiments had shown that many homeodomain containing transcription factors, including the Oct proteins, contain a DNA-binding domain which is resistant to digestion

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30 Figure 1. Purification of octamer binding proteins from mouse B-cell hybridoma. 100 ILI nuclear extract (- 1 mg protein) were incubated with 50 ng labelled oligonucleotide, 80 jig competitor DNA (poly dIdC) in 400 1I binding buffer (20 mM Hepes, 0.2 mM EDTA, pH 7.9, 200 mM NaCI, 15% glycerol). UV crosslinking was carried out as described (2). 20 Al aliquots from all column fractions containing radioactivity were analyzed on a 12% polyacrylamide gel. A. Autoradiograph, B. silver stained gel. Lane 1, molecular size marker; lanes 2-7, flow through and wash fractions; lanes 8-15, eluted fractions; lane 16, original sample. C. Aliquots of competition experiments made in the presence of 100 ng specific promoter oligonucleotide (lane 1) or 100 ng of a nonspecific oligonucleotide (lane 2).

4104 Nucleic Acids Research, Vol. 20, No. 15 with a variety of proteases (5). B-cell nuclear extract was digested with protease V8 before crosslinking to the promoter oligonucleotide. The protease treated sample was then purified over dT-cellulose as described above. After SDS - PAGE separation, a 30 kDa complex was blotted to glass fiber filter and submitted to microsequencing using the highly sensitive fluorescence detection method (3, 4). Starting with less than 1 pmol of purified V8 digested peptide - DNA complex, it was possible to determine 12 amino acid residues from the NH2-terminus with the following sequence: GXPGRRRKKR. Comparing this sequence to published cDNA sequences, we concluded that the protease-resistant domain starts at the amino terminus of the POU homeodomain of mOct-2. The method presented here thus allows to directly isolate DNA binding domains of transcription factors in sufficient purity for microsequencing.

ACKNOWLEDGEMENTS We thank A.Tsugita for many stimulating discussions and for carrying out the protein microsequencing, and M.Ostermayer and R.Ackermann for helpful comments on the manuscript. This project was supported by the Swiss National Science Foundation.

REFERENCES 1. Chodosh,L.A. et al. (1986) Cell 6, 4723-4733. 2. Brack,C. et al. (1988) J. Ultrastruct. Mol. Struct. Res. 101, 145-158.

3. Tsugita,A. et al. (1989) J. Biochem. 106, 60-65. 4. Tsugita,A. and Kamo,M. (1991) In Jornvall,H. et al. (eds), Methods in Protein Sequence Analysis. Birkhauser, Basel, pp. 123-132. 5. Brack,C. and Wichser,U. unpublished results.

Rapid isolation of specific DNA-binding proteins and their DNA-binding domains.

Nucleic Acids Research, Vol. 20, No. 15 4103-4104 . 1992 Oxford University Press Rapid isolation of specific DNA-binding proteins DNA-binding domain...
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