Protein DNA Interactions: Techniques Used

Mark A Strauch, University of Maryland, Baltimore, Maryland, USA

A plethora of biochemical and biophysical techniques are used to investigate various aspects of protein DNA interactions. Some of the more common and useful of these are filter binding, the gel mobility shift assay, a large group of related techniques broadly categorized as footprinting, high-resolution microscopy and spectroscopy.

Secondary article
Article Contents
. Introduction . Filter Binding . Gel Mobility Shift Assay . Footprinting . High-resolution Microscopy . Spectroscopic Approaches . Concluding Remarks

Introduction
The investigation of proteindeoxyribonucleic acid (DNA) interactions requires methods that can determine: (1) the strength, speed and stability of binding; (2) what determinants on the protein and DNA are interacting or otherwise needed for the interaction to occur; (3) what are the structural and physical relationships of the components in the initial, intermediate and nal stable congurations. This article briey surveys some of the more common means used to make these determinations. Detailed methodologies and more in depth background, theoretical and practical considerations, including discussion of relative advantages and disadvantages of the techniques, can be found in the references.

nique are that it is unable to discriminate between dierent types of bound complexes that may be formed, nor can it be easily adapted to study binding of multiple proteins to the same DNA molecule.

Gel Mobility Shift Assay

The gel mobility shift assay, also known as electrophoresis mobility shift assay (EMSA) or gel retardation, is one of
* x y P * x y * * y P:x * * *

x P:y

* * PP:xy *

Filter Binding
The oldest biochemical technique still in common use to study proteinDNA interactions is membrane ltration. Free nucleic acids will pass through nitrocellulose lters under a variety of nondenaturing buer conditions that lead to retention of proteins and proteinDNA complexes (Wong and Lohman, 1993). Although the physicochemical basis of this selectivity is not completely understood, and not all proteinDNA interactions are amenable to study by this technique, the methodology is usually quite rapid and reproducible (Figure 1a). Radioactive or other type of quantiably labelled DNA is mixed with the binding protein under suitable conditions and the mixture is passed through a lter. The amount of labelled DNA retained by the lter is a measure of the extent of the binding reaction that occurred before ltration. Data obtained can be used to derive equilibrium constants and rate constants, and to assess binding cooperativity by analysis using Scatchard or Hill plots. Additionally, if labelled DNA samples retained on the lter and present in the ltrate can both be recovered, this method of separation can be utilized in interference and missing-contact techniques to study the specic binding sites on the DNA (see below, Footprinting). Signicant disadvantages of the lter-binding tech-

UNB

Filter

PP:xy P:x,P:y

UNB (a) (b)

Gel

Figure 1 Filter binding and gel mobility shift assays. For illustrative purposes, a hypothetical set of interactions between a binding protein (P) and a DNA fragment containing two separate and independent binding sites (x and y) is shown. Separation of a mixture of unbound (UNB) DNA and each of the three possible bound forms, by (a) filter binding and (b) gel mobility shift, is depicted. Note that filter binding does not discriminate between the different bound forms, but the gel mobility shift is able to fractionate the doubly-bound complexes away from the singly-bound species (in this case it is assumed that the two distinct singly-bound species cannot be distinguished solely on the basis of gel mobility).

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Protein DNA Interactions: Techniques Used

the most common and powerful methods currently used to study proteinDNA interactions (Lane et al., 1992). As is the case with lter binding, the assay itself is relatively simple and takes advantage of dierent physical properties of proteinDNA complexes versus unbound DNA molecules in order to separate the distinct forms. A protein DNA binding reaction is subjected to electrophoresis through a gel matrix (usually polyacrylamide) and the gel is subsequently scanned for the positions and amounts of DNA present in bands of diering electrophoretic mobility (Figure 1b). If a small linear DNA fragment (typically less than 500 base pairs) or oligonucleotide is used, then the proteinDNA complex will usually migrate more slowly relative to the unbound DNA (hence the term gel retardation). The concentration of each species is usually determined by quantifying the amount of DNA present (most commonly the substrate DNA molecules have been radiolabelled). These values essentially represent the amounts of each DNA form present at the moment the binding mixture entered the gel. Data of this type can be used to derive equilibrium-binding constants (typically the dissociation constant, Kd, expressed in terms of protein concentration), kinetic parameters (association and dissociation rates) and assessments of binding cooperativity. The gel mobility shift assay combines a number of advantageous properties into a simple format when compared with many other proteinDNA binding assays. If the DNA fragment contains a very specic target site for the protein being studied, specic binding interactions can easily be detected even if the protein is present in a heterogenous mix containing other DNA-binding proteins (such as crude cellular extract). If a mixed population of DNA fragments, some having and others lacking a target site, is used in the assay, those fragments possessing active binding sites can be readily identied. The assay can often resolve DNAprotein complexes that dier only in protein to DNA stoichiometries (however, determination of absolute stoichiometries usually requires additional, more rigorous approaches). Complexes having the same stoichiometry but which dier in the physical arrangement or conformation of the components can be resolved in many cases. The method can be used to detect the binding behaviour of more than one type of protein to the same DNA molecule, provided the dierent complexes are easily distinguishable in relative mobility. (In some cases antibodies directed towards one of the protein components can be used to supershift the mobility of the complexes containing that binding protein.) Thus, the assay has the potential for studying thermodynamic and competitive properties of two or more proteins binding to the same DNA molecule, or specic site thereon. The gel mobility shift assay also forms the basis of a commonly used technique to assess bending of DNA. A permuted series of DNA fragments, all of equal length, are constructed so as to place the region to be studied (either sequences suspected to be intrinsically bent or a protein2

binding site) at dierent positions relative to the ends of the fragments. It has been observed that having a DNA bend near the centre of a fragment will result in less mobility than if the bend is close to an end of the fragment. Proteininduced DNA bending is manifested by slower migration of complexes in which the protein is bound near the middle of the DNA fragment, as opposed to having the protein bound near an end of the DNA.

Footprinting
Footprinting is a broadly dened term encompassing a large group of related techniques used to study protein DNA interactions (it should not be confused with the term DNA ngerprinting). The commonality between DNA footprinting techniques is that they are all in some way used to elucidate the sequence(s) that either comprise, or are in close proximity to, the nucleotide moieties actually interacting with the bound protein. The various techniques can be further subdivided into two broad categories: protection assays and interference assays. (Details concerning footprinting reagents and methods mentioned in this section, as well as extensive listings of original references, can be found in the methodology manuals listed at the end of this article.)

Protection assays
The principle behind the protection category of DNA footprinting assays is that binding of a protein to DNA will hinder access of a nucleolytic or chemical modication agent to positions of the DNA helix that are interacting with, or in some way altered by, the binding protein (Figure 2). The proteinDNA complexes are subjected to limited endonuclease cleavage or to limited chemical modication and cleavage. The population of DNA fragments of varying length (usually previously labelled with a radioisotope) are fractionated by denaturing gel electrophoresis (such as the type used for DNA sequencing). Side-by-side comparison of the cleavage pattern of proteinDNA complexes versus unbound DNA is made. Many base pair positions in the region bound by the protein usually exhibit some degree of protection from attack relative to the same region in the control reaction using unbound DNA. The precise locations of these positions are determined by reference to chemical sequencing (Maxam-Gilbert DNA sequencing) ladders produced from separate aliquots of the same labelled DNA fragment. Often, positions which become more susceptible to cleavage or modications are observed in these assays. Observations of such hypersensitive positions, which relate to structural deformation of the DNA induced by protein binding, can be as informative as observing protected positions.

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Protein DNA Interactions: Techniques Used

+ Binding protein *

+ DNAase I

+ DNAase I

Footprint

Figure 2 DNAase I footprinting: an example of the protection class of footprinting assays. Unbound and protein-bound DNA fragments (labelled (asterisk) at one end on one strand) are treated with limiting amounts of DNAase I (no more than one cleavage per molecule). The digestion reactions are fractionated on a denaturing polyacrylamide gel. Protein bound to DNA protects areas from DNAase I attack. The absence of bands corresponding to digestion products generated by cleavage in these areas defines the protein-binding site (the footprint).

The most common endonuclease used in footprinting is DNAase I but others with relatively broad specicity, such as micrococcal nuclease, can be employed. Exonuclease III of Escherichia coli, which processively digests in a 3! 5 direction only a single strand of duplex DNA, has also been used to identify approximate endpoints of protein-binding regions. Unfortunately, DNAase I and other common endonucleases do not cleave DNA in a totally random fashion. While some sequences are readily cleaved, others are very recalcitrant to digestion, even after prolonged incubation with the enzyme. This, along with the relatively large size of these enzymes, limits the resolution of the technique. Better resolution can usually be obtained by using small reactive chemical species that cleave DNA in a more sequenceindependent manner. Additionally, the small size of these agents permits them to gain access to exposed regions in proteinDNA complexes that are sterically inaccessible to nucleases. Two of the more useful and widely used of these chemical nucleases are hydroxyl radicals (generated by the Fenton reaction) and 1,10-phenanthroline-copper, both of which cleave DNA by attacking deoxyribose moieties. Other agents that target DNA in a more base-specic manner can be used for ne-structure footprinting. Dimethyl sulfate (DMS), which methylates purine bases (primarily the N7 position of guanines), is commonly used. Potassium permanganate (KMnO4) is a strong oxidant

that reacts with base moieties of DNA but exhibits the strongest reactivity towards single-stranded or unpaired bases, primarily thymines. KMnO4 has been used to great eect in detecting localized helix melting (as during formation of open transcription complexes by ribonucleic acid (RNA) polymerase) and for detecting other structural transitions of DNA that accompany protein binding. Osmium tetroxide also shows specicity for unpaired or unstacked thymines and can sometimes be used if buer conditions inhibit KMnO4 probing. Diethylpyrocarbonate (DEPC) carbethoxylates N7 positions of unstacked purine bases and is another agent used to detect alterations 1 due to protein binding. Uranyl (VI) ions (UO2 2 ) bind to the phosphate backbone and will lead to cleavage of DNA when photochemically excited by light in the range of 300 1 photofootprinting is a sensi420-nm wavelengths. UO2 2 tive probe for proteinDNA phosphate contacts as well as conformational changes aecting phosphate accessibility (minor groove width). Another type of photofootprinting simply utilizes ultraviolet light to probe availability of bases to form photoproducts (primarily dimers between adjacent residues). In cases where the agent (notably DMS, KMnO4, ultraviolet light) can readily enter intact cells, in vivo footprinting can be performed. The cells are exposed to the agent, followed by isolation of the chromosomal DNA. Primer extension methods are used to label fragments from the region of interest. These fragments will be a population having the same 5 endpoint but diering in 3 endpoints, the latter corresponding to the dierent sites of previous modications or cleavage. This series of fragments can then be fractionated on denaturing acrylamide gels and analysed, just as is done for in vitro footprinting. One potential pitfall in the use of many of these physical and chemical footprinting agents is that they can also be reactive towards entities in the binding protein being studied. However, if reaction conditions are carefully chosen and controlled, this rarely will present a signicant problem.

Interference assays
Footprinting interference assays are the inverse of the protection assays, in that the chemical modication of the DNA target is performed before exposure to the binding protein. The two most common modications used in interference assays are methylation at N7 positions of guanines (via DMS, see above) and ethylation of the phosphate backbone (via ethylnitrosourea). After limited modication treatment, the DNA is combined with the binding protein and the mixture separated into bound and unbound fractions (e.g. via gel mobility shift or lter binding). The separate fractions are then subjected to an appropriate treatment that will cleave the DNA strand adjacent to the site of the modication. These separate
3

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Protein DNA Interactions: Techniques Used

populations of fragments are then run side-by-side on a denaturing acrylamide gel. If modication at a certain position inhibited binding by the protein, then a band corresponding to that position will be prominent in the unbound fraction lane but will be either absent or diminished in the bound fraction lane (Figure 3). In cases where excision of a single base or nucleoside moiety from an otherwise intact double-stranded helix occurs, these interference assays are also known as missing contact assays. The most frequently used reaction chemistries are random depurination (Maxam-Gilbert G 1 A chemical sequencing reaction), depyrimidination (Maxam-Gilbert C 1 T reaction) or random excision via hydroxyl radical attack (see above). Subsequent protein binding, separation of bound from unbound fractions and detection of important bases is analogous to that described above. The observation that a missing nucleoside leads to inhibition of protein binding should not automatically be construed as evidence that the protein normally makes
Binding site * Limited modification (1) * (2) *

contact with that residue. Removal of a nucleoside by these procedures alters a number of DNA structural parameters: base stacking forces are disrupted and constraints on localized helical parameters are perturbed, as the chemical reactions employed also nick the phosphodiester backbone of one strand. Conversely, if the DNA fragment being examined contains two or more independent binding sites not able to be discriminated by the method used to separate bound from unbound DNA, then loss of a contact in one site will not be manifest as a loss of binding to the fragment as a whole. Nevertheless, when combined with results from other types of binding assays, missing contact experiments usually provide valuable data regarding proteinDNA interactions.

Proteolytic footprinting
Biochemical probing of proteinDNA interactions can also be investigated by determining how protein reactivity changes upon binding to a DNA target. Analogous to footprinting of DNA, proteases or chemical cleavage agents can be used to examine protein regions that are aected (protection from cleavage; hyperreactivity) upon DNA binding (Bogenhagen, 1993; Heyduk and Heyduk, 1994). These assays are more technically demanding than DNA footprinting, in that they entail the need to separate and analyse proteolytic peptides. Protection and interference assays based upon modication of amino acid residues can also be performed to identify protein moieties involved in binding. Although still relatively in its infancy, methodological advances may someday make proteolytic footprinting as commonplace in laboratories as DNAase I footprinting.

(3) *

(4) * + Binding protein B Gel mobility shift UNB Excise bands, cleave UNB 3 2 1 B 4 Denaturing gel

High-resolution Microscopy
Transmission electron microscopy has been used to characterize multiproteinDNA complexes that are either too large or too complex to be readily analysed solely by biochemical means (Thresher and Grith, 1992). Examples include complexes involved in DNA replication, RNA transcription, DNA recombination and chromatin structure. Coupled with immunoelectron methodology, individual protein components and nucleoprotein reaction intermediates in such complexes can be examined. ProteinDNA interactions also have been visualized by scanning tunnelling microscopy, but the method has yet to be widely used for these applications. Cryoelectron microscopy, coupled with sophisticated image analysis and molecular modelling techniques, potentially can reveal structural details of large and complex nucleoprotein assemblies at signicant resolutions (Darst et al., 1998). The recent development of the scanning force microscope (also called atomic force microscope) has provided

Figure 3 Footprinting interference assay. End-labelled DNA (asterisk) is subjected to limited chemical modification before exposure to the binding protein. The binding reaction is fractionated via a gel mobility shift assay to separate bound (B) from unbound (UNB) species. The DNA in the bound (retarded) band will either be unmodified or contain modifications at positions not affecting binding. The DNA in the unbound band will be comprised of molecules in which modification has occurred at positions affecting interaction with the protein. The DNA from both bands is isolated, subjected to strand cleavage at positions adjacent to the site of modification, and fractionated on a denaturing polyacrylamide gel. Positions which, when modified, affect protein binding will produce corresponding cleavage products only in the sample from the unbound species.

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Protein DNA Interactions: Techniques Used

an exciting and powerful new means of studying protein DNA interactions. A major attraction is that, unlike other methods, scanning force microscopy can provide nanometer resolution under physiological conditions (Bustamante and Rivetti, 1996).

Concluding Remarks
A large arsenal of methods is available to pry information out of nature regarding proteinDNA interactions. New methods are continually being developed and older methods being rened. Detailed investigations require multipronged approaches, with each dierent technique providing unique information. The brief discussion given here is not intended to be all-inclusive: it should not be construed as meaning that methods not mentioned are intellectually less desirable or are unable to provide signicant insights.

Spectroscopic Approaches
A number of spectroscopic methods, used to investigate the nature and structure of proteins and DNA individually, have been adapted to study proteinDNA interactions. Changes in a protein or DNA emission or absorption spectrum as it participates in a binding interaction provides a means of following the reaction and generates data concerning conformational changes that occur. Fluorescence spectroscopic methods are relatively sensitive and usually easy to perform, and can measure true binding equilibria and stoichiometries. Often, the intrinsic uorescence of a binding protein (due to its tryptophan and tyrosine residues) is altered upon complex formation with DNA. Of course, not all binding proteins have tryptophan or tyrosine residues positioned to serve as sensitive indicators of structural changes. In some cases it may be possible to attach covalently a uorescent probe to the protein or to engineer genetically a tryptophan residue into a position of the protein that, while not perturbing binding, can serve as a probe of conformational changes. Alternatively, use might be made of a type of binding competition assay using as probe a uorescent ligand that is displaced from the protein (or DNA) upon complex formation (Taylor and Kneale, 1994). Recent advances in the development of uorescent probes and the ability to incorporate uorescent base analogues into synthetic oligonucleotides have led to an expanded ability to use uorescence measurements to study proteinDNA interactions (Millar, 1996). Rapid technological advances are also being made in the use of Raman spectroscopy to study nucleoprotein complexes (Thomas, 1999). Finally, the development of devices (biosensors) based upon the phenomenon of surface plasmon resonance have provided researchers with a new and sophisticated means of studying the equilibria and kinetics of proteinDNA interactions in real time (Schuck, 1997). Other spectroscopic methods can be used to gain additional or conrmatory evidence of certain aspects of proteinDNA interactions. For example, circular dichroism has been used to assess binding stoichiometries and show that the conformation of a protein bound at a specic target site diers from its conformation when bound nonspecically to DNA. Of course, mass spectrometry, multidimensional nuclear magnetic resonance and X-ray crystallography provide ultimate structural details of stable proteinDNA complexes.

References
Bogenhagen DF (1993) Proteolytic footprinting of transcription factor TFIIIA reveals dierent tightly binding sites for 5S RNA and 5S DNA. Molecular and Cellular Biology 13: 51495158. Bustamante C and Rivetti C (1996) Visualizing proteinnucleic acid interactions on a large scale with the scanning force microscope. Annual Review of Biophysics and Biomolecular Structure 25: 395429. Darst SA, Polyakov A, Richter C and Zhang G (1998) Structural studies of Escherichia coli RNA polymerase. Cold Spring Harbor Symposia on Quantitative Biology 63: 269276 Heyduk E and Heyduk T (1994) Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach. Biochemistry 33: 96439650. Lane D, Prentki P and Chandler M (1992) Use of gel retardation to analyze proteinnucleic acid interactions. Microbiological Reviews 56: 509528. Miller DP (1996) Fluorescence studies of DNA and RNA structure and dynamics. Current Opinion in Structural Biology 6: 322326. Schuck P (1997) Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of the interactions between biological macromolecules. Annual Review of Biophysics and Biomolecular Structure 26: 541566. Taylor I and Kneale GG (1994) A competition assay for DNA binding using the uorescent probe ANS. In: Kneale GG (ed.) DNA Protein Interactions: Principles and Protocols, pp. 327337. Totowa, NJ: Humana Press. Thomas GJ Jr (1999) Raman spectroscopy of protein and nucleic acid assemblies. Annual Review of Biophysics and Biomolecular Structure 28: 127. Thresher R and Grith J (1992) Electron microscopic visualization of DNA and DNAprotein complexes as adjunct to biochemical studies. Methods in Enzymology 211: 481490. Wong I and Lohman TM (1993) A double lter method for nitrocellulose-lter binding: application to proteinnucleic acid interactions. Proceedings of the National Academy of Sciences of the USA 90: 54285432.

Further Reading
Jost JP and Saluz HP (eds) (1991) A Laboratory Guide to In Vitro Studies of Protein DNA Interactions. Basel: Birkha user. Kneale GG (ed.) (1994) DNA Protein Interactions: Principles and Protocols. Totowa, NJ: Humana Press. Revzin A (ed.) (1993) Footprinting of Nucleic Acid Protein Complexes. San Diego: Academic Press. Sauer RT (ed.) (1991) ProteinDNA interactions. Methods in Enzymology 208.

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