Methylation and Uracil Interference Assays for Analysis of Protein‐DNA Interactions

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Interference assays identify specific residues in the DNA binding site that, when modified, interfere with binding of the protein. The protocols use end?labeled DNA probes that are modified at an average of one site per molecule of probe. These probes are incubated with the protein of interest, and protein?DNA complexes are separated from free probe by the mobility shift assay. A DNA probe that is modified at a position that interferes with binding will not be retarded in this assay; thus, the specific protein?DNA complex is depleted for DNA that contains modifications on bases important for binding. After gel purification, the bound and unbound DNA are specifically cleaved at the modified residues and the resulting products analyzed by electrophoresis on polyacrylamide sequencing gels and autoradiography. In the methylation interference protocol presented here, probes are generated by methylating guanines (at the N?7 position) and adenines (at the N?3 position) with DMS; these methylated bases are cleaved specifically by piperidine. In the uracil interference protocol, probes are generated by PCR amplification in the presence of a mixture of TTP and dUTP, thereby producing products in which thymine residues are replaced by deoxyuracil residues (which contains hydrogen in place of the thymine 5?methyl group). Uracil bases are specifically cleaved by uracil?N?glycosylase to generate apyrimidinic sites that are susceptible to piperidine. These procedures provide complementary information about the nucleotides involved in protein?DNA interactions.

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Basic Protocol 1: Methylation Interference Assay
Basic Protocol 2: Uracil Interference Assay
Reagents and Solutions
Commentary
Figures

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Materials

Basic Protocol 1: Methylation Interference Assay

Materials

DNA containing protein‐binding site
TE buffer, pH 7.5 to 8.0 ( appendix 22 )
Dimethyl sulfate (DMS)
recipeDMS reaction buffer
recipeDMS stop buffer
10 mg/ml tRNA solution
0.3 M sodium acetate/1 mM EDTA, pH 5.2
1 M piperidine (dilute from 10 M piperidine stock)
Stop/loading dye (unit 7.4 )
90° to 95°C water bath

Additional reagents and materials for mobility shift DNA‐binding assay (unit 12.2 ), phenol extraction and ethanol precipitation (unit 2.1 ), nondenaturing polyacrylamide gel electrophoresis and electroelution of DNA fragments onto DEAE membranes (unit 2.7 ), autoradiography ( appendix 3A ), and denaturing polyacrylamide gel electrophoresis for sequencing (unit 7.6 )

Basic Protocol 2: Uracil Interference Assay

Materials

DNA containing protein‐binding site
Oligonucleotide primers specific for sequences flanking the binding site on the two complementary DNA strands
2 mM 4dNTP mix (unit 3.4 )
0.5 mM dUTP
Taq polymerase buffer (unit 3.4 ) and Taq polymerase (unit 3.5 )
TE buffer, pH 7.5 to 8.0 ( appendix 22 )
Uracil‐N ‐glycosylase (Perkin Elmer‐Cetus)
1 M piperidine (diluted from 10 M piperidine stock)
Stop/loading dye (unit 7.4 )
90° to 95°C water bath

Additional reagents and equipment for end‐labeling DNA with T4 polynucleotide kinase (unit 3.9 ), polymerase chain reaction (unit 15.1 ), phenol extraction and ethanol precipitation (unit 2.1 ), nondenaturing polyacrylamide gel electrophoresis and electroelution of DNA onto DEAE membranes (unit 2.7 ), denaturing polyacrylamide gel electrophoresis (unit 7.6 ), mobility shift DNA‐binding assay (unit 12.2 ), and autoradiography ( appendix 3A )

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Figures

Figure 12.3.1 Uracil interference. An oligonucleotide or restriction fragment (long arrow) containing a protein‐binding site (hatched box) is amplified by PCR using one unlabeled primer (short arrow) and one 5′‐labeled primer (asterisk) in the presence of dGTP, dATP, dCTP, dTTP, and dUTP, producing reaction products in which deoxyuracil is randomly substituted for thymine on both strands. This collection of DNA molecules is incubated with the protein of interest and DNA molecules containing deoxyuracil substitutions that do not interfere with protein binding are selected by purifying the DNA‐protein complex away from unbound DNA. The resulting DNA is cleaved at uracil residues using uracil‐ N ‐glycosylase followed by piperidine, and the reaction products are separated on a denaturing polyacrylamide gel. (Reprinted from Pu and Struhl, , by permission of Oxford University Press.)

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Figure 12.3.2 Hypothetical autoradiogram of a methylation interference experiment, as described in Anticipated Results. Guanines and adenines that interfere are indicated by asterisks.

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Literature Cited

Literature Cited
	Baldwin, A. and Sharp, P. 1988. Two transcription factors, H2TF1 and NF‐kB, interact with a single regulatory sequence in the class I MHC promoter. Proc. Natl. Acad. Sci. U.S.A. 85:723‐727.
	Brunelle, A. and Schleif, R. 1987. Missing contact probing of DNA‐protein interactions. Proc. Natl. Acad. Sci. U.S.A. 84:6673‐6676.
	Goeddel, D.V., Yansura, D.G., and Caruthers, M.H. 1978. How lac repressor recognizes lac operator. Proc. Natl. Acad. Sci. U.S.A. 75:3579‐3582.
	Ivarie, R. 1987. Thymine methyls and DNA‐protein interactions. Nucl. Acids Res. 15:9975‐9983.
	Maxam, A. and Gilbert, W. 1980. Sequencing end‐labeled DNA with base‐specific chemical cleavages. Methods Enzymol. 65:499‐560.
	Pu, W.T. and Struhl, K. 1992. Uracil interference, a rapid and general method for defining protein‐DNA interactions involving the 5‐methyl group of thymines: The GCN4‐DNA complex. Nucl. Acids Res. 20:771‐775.
	Siebenlist, U. and Gilbert, W. 1980. Contacts between E. coli RNA polymerase and an early promoter of phage T7. Proc. Natl. Acad. Sci. U.S.A. 77:122‐126.
Key References
	Hendrickson, W. and Schleif, R. 1985. A dimer of AraC protein contacts three adjacent major groove regions at the Ara I DNA site. Proc. Natl. Acad. Sci. U.S.A. 82:3129‐3133.
	Provides good descriptions of methylation and ethylation interference procedures as well as interpretations of major groove contacts.
	Maxam and Gilbert 1980. See above.
	Describes DNA labeling, modification by DMS, and polyacrylamide gel electrophoresis.
	Pu and Struhl, 1992. See above.
	Describes original method for uracil interference.