Chemical and photochemical probing of DNA complexes.

REVIEW PAPER

Chemical and Photochemical Probing of DNA Complexes Peter E. Nielsen Research Center for Medical Biotechnology, Department of Biochemistry B, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark An overview of the chemical and photochemical probes which over the past ten years have been used in studies of DNA/ligand complexes and of non-Bform DNA conformations is presented with emphasis on the chemical reactions of the probes with DNA and on their present ‘use-profile’. The chemical probes include: dmethyl sulfate, ethyl nitroso urea, diethyl pyrocarbonate, osmium tetroxide, permanganate, aldehydes, methidiumpropyl-EDTA-Fell (MPE), phenanthroline metal complexes and EDTA/FeII. The photochemical probes that have been used include: psoralens, UVB, acridines and uranyl salts. The biological systems analysed by use of these probes are reviewed by tabulation.

1. INTRODUCTION

Following the discovery of the double helical DNA structure (Watson and Crick, 1953) and the subsequent determination of a rapidly increasing number of DNA sequences, has come a need to understand how the linear DNA macromolecule interacts with other molecules of the cell, and of the environment, in the course of genetic function. Numerous proteins interact with DNA either as structural elements, exemplified by the histones of eukaryotic chromatin or the HU proteins of prokaryotic cells, or as members of the complex cellular machinery involved in gene expression and DNA replication. Histone (and HU) DNA interactions are largely sequence neutral and rely mostly on electrostatic binding to the phosphate backbone, whereas the DNA binding of, e.g., transcription factors is highly sequence-dependent. Such sequence discrimination by proteins involves specific hydrogen bonding between amino acid residues of the protein and the purines and pyrimidines of the DNA helix as well as Van der Waals’ and electrostatic interactions (see Ollis and White, 1987, for a recent review on protein/DNA interactions). Sequence dependent binding to DNA has also been observed with many drugs, and this sequence specificity most likely relates to their biological activity. The last 10 years of research have, furthermore, made it clear that DNA can adopt biologically relevant conformations other than B-DNA (e.g., A-, Z- and H-DNA, cruciforms, etc.). Even the common B-DNA exhibits significant microheterogeneity in terms of base twist, roll, tilt angles and base rise and slide, etc. (see Dickerson, 1989, for the definitions of these DNA parameters). Although the ultimate detailed molecular description of DNA complexes requires x-ray and/or ‘H NMR studies, the development and application of a veritable armoury of enzymatic, chemical and photochemical probes has made it possible to obtain quite detailed

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molecular descriptions of a large number of DNA complexes ranging from chromatin fibers via polymerase and transcription factor/DNA complexes to low molecular drug/DNA complexes. The use of probes, furthermore, allows for dynamic investigations as well as studies in situ and in vivo. The intention of this paper is to give an overview of the chemical and photochemical probes that have been employed within the past 10 years in studies of DNA complexes, including non-B-DNA conformations. Depending on the particular probe and the experimental set-up, various kinds of information about the DNA complex can be obtained. In the traditional footprinting approach (Fig. I), the difference in DNA reactivity towards the probe with or without bound ligand, is analysed. Decreased reactivity is interpreted as ligand/DNA contacts (proximity), whereas enhanced reactivity usually indicates a change in DNA conformation. In an interference analysis, the DNA is first /

binding silo

PAGE

Figure 1. Principle in a footprinting experiment. The DNA molecule . cleaved (t) at a density is labeled on one strand at one end ( 0 ) and of one cleavage per DNA molecule. Each cleavage point thus corresponds to a specific length of labeled DNA fragment as analysed by gel electrophoresis. A, control; 6,in the presence of protein (ligand).

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reacted with the probe and the population of modified DNA molecules that have retained ligand binding capacity is isolated (usually by filter binding or gel electrophoresis band shift procedures) and analysed. This method identifies regions and features of the DNA that are essential for ligand/DNA complex formation. An inspection of the B-DNA structure (Fig. 2) reveals that chemical or photochemical probing can take place at any one of the three ‘functional moieties’ of the DNA: the phosphates, the deoxyriboses or the bases. Reactive positions of the bases in double stranded DNA are guanine (and adenine) N7 in the major groove (Fig. 2)

and adenine N3 in the minor groove, and the reactivity of these positions is absent if ligands are bound here. The 5,6 double bond of thymine is, furthermore, able to engage in cycloaddition reactions. The phosphatedeoxyribose backbone of the DNA is not particularly shielded from probes, but the relative accessibility is of course influenced by ligands contacting the backbone and also to a lesser extent by the DNA conformation in terms of phosphate-phosphate distances (‘groove size’) and deoxyribose conformations (e.g., puckering). The reactivity of the bases, on the other hand, is highly sensitive to changes in DNA conformation since the bases are ‘hidden’ in the center of the helix. The base pairing positions in particular are totally shielded in double stranded DNA and reaction at these positions indicates breakage of hydrogen bonding. The majority of the probes exploited so far and discussed in this review are based on the footprinting/ interference principle and thus analyse DNA/ligand complexes in terms of the DNA, but a few techniques are aimed at the ligand part (in these cases proteins) of the complex. These techniques involve chemical or photochemical crosslinking of the complex which allows for the isolation and subsequent identification (usually by immunochemical or radioisotopic means) of the DNA bound proteins.

2. CHEMICAL PROBING 2.1 Attack on DNA bases Dimethyl sulfate. Dimethyl sulfate (DMS, 1) methylates N7 of guanine and N3 of adenine (Fig. 2) in double stranded B-form DNA (Lawley and Brookes, 1963). In single stranded DNA, N1 of adenine and to a lesser extent N3 of cytosine are also susceptible to methylation (Lawley, 1966). These are involved in hydrogen bonding in double stranded DNA (Fig. 2).

2.1.1.

w

CH3-O-S-O-

CH,

b 1

H‘

m

GUANINE

CYTOSINE M

n. CH 1

3

rn

ADENINE

THYMINE

Figure 2. 6 - D N A model molecular graphics of the Drew-Dickerson 12-mer (Wing ef a/., 1980); with, below, G-C and A-T Watson-Crick base pairs. M, major groove; m, minor groove. All molecular graphics were performed on a Silicon Graphics ‘Personal Iris’ using the Biosym programmes ’Insight’ and ‘Discover‘.

2 JOURNALOF MOLECULAR RECOGNITION, VOL.3, No. 1,1990

The DMS methylation reaction of guanine N7 in the DNA major groove (Fig. 3) has been used extensively for both footprinting and interference studies of ligandl DNA complexes as pioneered by Gilbert and coworkers. The analysis takes advantage of the labilization of the base-sugar glucosylic .bond upon alkylation of purine N7, which by analogy with the chemical DNA sequence reaction allows specific base catalysed strand scission at the alkylated purine (Maxam and Gilbert, 1977). A DMS footprinting experiment thus probes the accessibility of guanine N7 (Fig. 3) and can identify ligand/guanine contacts in the major groove (Gilbert et al., 1976). Numerous protein/DNA and drug/DNA complexes have been analysed by this technique (cf. Table l), and recently the methylation protection technique has also been applied for in vivo studies (Nick and Gilbert, 1985) exploiting the genome sequencing proto-


CHEMICAL A N D PHOTOCHEMICAL PROBING OF DNA COMPLEXES

relative populations of possible tautomeric forms of the DNA base, and in this way the stability of the base pair.

2

Diethyl pyrocarbonate (DEPC, 3) reacts with N7 of purines in DNA resulting in the ring-opened product 4 (Scheme 1) (Vincze et al., 1973). Due to this carbethoxylation and ring opening, the N9 glycosylic bond is labilized and treatment with alkali results in strand scission at the modified purine. In contrast to dimethyl sulfate, diethyl pyrocarbonate does not react very efficiently with double stranded B-DNA probably due to the size of the reagent. Guanine N7 in the left handed Z-DNA is considerably more exposed for carbethoxylation and consequently DEPC can be used to monitor the B-DNA-Z-DNA transition (Herr, 1985; Johnston and Rich, 1985; Vogt er al., 1988). DEPC reacts efficiently with purines in single stranded DNA and can, therefore, also be used to detect the ‘eye’ of cruciform hairpins (Scholten and Nordheim, 1986; Furlong and Lilley, 1986). Bis-intercalative binding of drugs to DNA also induces DEPC hyperreactivity. In the case of echinomycin this observation was originally interpreted in terms of formation of Hoogsteen base pairing exposing N3 of guanine and adenine in the major groove (Mendel and Dervan, 1987), but more extensive studies (McLean and Waring, 1988; Jeppesen and Nielsen, 1988; Portugal et af.,1988) supported by NMR results (Gao and Patel, 1988; Gilbert er af.,1989) conclude that the effect is more likely due to the intercalation induced unwinding of the 2.1.2 Diethyl pyrocarbonate.

Figure 3. Model of B-DNA modified with dimethyl sulfate guanine (N7, yellow), diethyl pyrocarbonate at guanine (N7, red), KMn04 ) (5.6 bond, green) or ethylnitrosourea (or (or 0 ~ 0at~ thymine phosphate, cyan) (the two isomers are shown).

col (Church and Gilbert, 1984) in which the DNA fragments are visualized by hybridization. Guanine N7 methylation is also ideally suited for interference studies. In this case guanines, which after methylation interfere with ligand binding and therefore contribute significantly to the DNA/ligand contacts, are identified (Siebenlist and Gilbert, 1980). Methylation of the base pairing positions N1 of adenine and N3 of cytosine is used to probe for regions of single stranded DNA as found, e.g., in the RNA polymerase promoter open complex (Siebenlist, 1979; Kirkegaard et af.,1983). Dimethyl sulfate is the classical chemical probe for studying ligand/DNA interactions, and results from a large variety of systems have proved its utility both using methylation protection and methylation interference assays. Upon interpreting the results, one should nevertheless keep in mind that DMS also methylates proteins and other ligands when these contain nucleophilic groups (-NH2, -SH, etc.) and may in this way perturb the complex. DMS is, furthermore, lipophilic and thus only sparsely soluble in water, and may because of this concentrate in ‘lipophilic pockets’ of the proteins. Methylation interference results are mostly interpreted in terms of steric interference by the methyl group at guanine N7, but one should consider that a positive charge that may influence ligand/DNA interaction has also been introduced at this position (N7-methyl guanosine = 2). The methylation may also change the

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e

o

CH3-CHrO-C-O-C-O-

CH2-CH3

3

4

Scheme 1

DNA helix. The details of the structural changes induced by intercalation that renders the DNA accessible to DEPC reaction is, however, still unknown. Recently, DEPC has been used for studying protein/ DNA interactions both by the interference (Heuer and JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 1,1990 3

P.E. NIELSEN

Table 1. Examples of systems analysed with chemical and photochemical probes Reagent

Site of attack on DNA

DMS

N7 of guanine

Probing for

guanine contact in major groove

System analysed

repressors

transcription factors etc.

recA

RNA polymerase EcoRl TFlllA

N3 of adenine N1 of cytosine

DEPC

N7 of guanine/adenine

N7-G which are pertinent for binding (interference) adenine contact in minor groove single strands

recombinase nucleosomes virus antibiotics 2-DNA, etc. repressors transcription factors repressor RNA polymerase

changes in DNA helix parameters (base twist angles?), single strands

Z-DNA

A/G major groove contacts

repressors

cruciform, etc. echinomycin/intercalators RNA polymerase

H-DNA"

Reference

44, 115,143, 145, 151, 195, 197, 198, 227, 239, 240, 278, 285,287,352 5, 19, 20, 53, 63, 124, 126, 157,172,221,222,223,256, 257,283,338,382,384,391 80,183 1.7.45. 145, 146, 163,316, 339 17 91 16 204 77, 165,217,219, 310, 349 99,208 367,369 143 3, 72 44143,239,240 34,169, 314,331

139, 164, 174, 205, 226, 276, 277,367 104, 194,291 97,98,160,208,211,258 13,34 255,370 143

(interference)

KMnO,

thymine 5.6 double bond changes in DNA helix parameters (major groove) (base twist angles, stacking?)

RNA polymerase echinomycin/intercalators DNA loops H-DNA, etc. 2-DNA

oso,

thymine 5.6 double bond changes in DNA helix parameters (major groove) (base twist angles stacking)

echinomycin promoter

base pair mismatch

207,208 6,347 107, 164,248; 249, 251, 364, 367 105, 123, 187,188, 194, 202, 203 119,255,368 70, 71

Z-DNA single strands

162,237,286 98,160 26 194,249 366

cruciform/hairpin DNA

H-DNA

ENU

phosphates

phosphates which are essential for binding (interference)

repressors recA RNA polymerase EcoRl resolvase virus/phage proteins

41,44, 79, 143,152, 241 183 316 17,350 269 165, 391

EDTA/FelI

deoxyribose

DNA backbone contacts/backbone conformation

repressors, etc. RNA polymerase resolvase hormone receptor

44,152,338,356 237,269 134 54 354,357,371 37 65 355 259

TFlllA bent DNA Holliday struct. nucleosomes distamycin

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CHEMICAL AND PHOTOCHEMICAL PROBING OF DNA COMPLEXES

Table 1. Examples of systems analysed with chemical and photochemical probes (continued) Reagent

MPE/Fell

Site of attack on DNA

deoxyribose

deoxyribose

Probing for

System analysed

intercalation interference (minor groove)

intercalation interference/changes in DNA helix parameters

Reference

lac-repressor RNA polymerase intercalators/minor groove binders misc. transcription factors, etc. chromatin

361 13,55,127,153,290 133, 142, 359, 360, 361

RNA polymerase promoters

1,40,86, 179, 330, 332, 333

repressors termination factor, etc.

179, 316 180,192

cruciform/hairpin

Z- D NA

105,123,173,386 174.248

134, 177, 288, 362 46, 47, 48

Bromacetaldehyde/ Glyoxal

adenine N1, N6

Formaldehyde

DNA/protein crosslinking protein/DNA proximity

chromatin

120,156,293,328,329

Depurination

DNA/protein crosslinking protein/DNA proximity

RNA polymerase nucleosomes

61 14, 88, 186, 265, 313, 321, 343

Psoralens

thymines (preferentially at 5'-TA sites)

chromatin

49, 50, 67, 69, 75, 130, 167, 193, 216, 245, 246, 261, 272, 300,326,351,379,381 266,301,327 73,307,311,312,325 301 171, 323 393,396 31 1,312,396 128,380,385 51,305 112

non- hydrogen bonded adenine

intercalation interference

replication complex transcription complex recombination 2-DNA repressors RNA polymerase phage packaging cruciform/hairpin hybridization

double strandedness

Acridines (azido/diazo)

DNA bases (deoxyriboses?)

intercalation interference

UVB

thymine (dimers) pyrimidine-pyrimidone (64-adducts) + ?

changes in DNA protein contacts

RNA polymerase echinomycin/minor groove binders

158 161

lac-repressor etc.

18,35 8,297,298,299

GAL4 activator and/or changes in DNA helix parameters

protein/DNA crosslinking protein/DNA proximity (thymine/lysine?)

TFlllA EcoR I nucleosomes chromatin virus RNA polymerase topoisomerase, etc.

UVBIBUdR

BUdR photochemistry at thymine protein proximity thymine positions and crosslinking to protein

UO22+

deoxyri boses

backbone phosphate contacts

lac-repressor RNA polymerase chromatin phagdvirus h-repressor RNA polymerase bent DNA TFlllA

373 17 109,110,373 2, 4, 9, 43, 76, 108, 178, 196. 200,292 57, 58, 59, 147, 236, 302 116, 117, 132, 144, 159, 252, 253,254,324.389 118,270,303 10,196,238 241, 319, 320 24.96,348 27,380 162,232 162 -b -b

a Conformation in some homopurin-homopyrimidine sequences involving a combined triple helix/single strand DNA form, by some referred to as H-DNA (Mirkin etal., 19871. Jeppesen and Nielsen (in prepaiation).


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RECOGNITION,VOL. 3, No. 1,1990 5

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Hillen, 1988) and the hypersensitivity (Bateman and Paule, 1988; Buckle and Buc, 1989) principle. The latter type of studies should be interpreted with great caution, however, since, as also demonstrated by Buckle and Buc (1989), DEPC (Boehm and Metha, 1938) reacts very efficiently with protein (Sams and Mathews, 1988). In fact, DEPC was originally introduced as a reagent for protein modification (Miller, 1977). 2.1.3 Osmium tetroxide (OsO,). Osmium tetroxide (OsO,)

is a well known oxidation reagent for alkenes in organic chemistry. Likewise thymines and, to a much lesser extent, cytosines in DNA are oxidized to 5,6 cis-diols by OsO, via stable OsO, adducts (Scheme 2) (Beer et al., 1966; Dizdaroglu et al., 1986).

Scheme 2

Double stranded B-DNA is relatively resistant to OsO, thymine oxidation due to the stacking of the bases which partially shields the thymine 5,6 double bond. (It is to be recalled that oxidation takes place from the face of the aromatic ring.) OsO, oxidation is enhanced if the DNA is in a single stranded conformation, e.g., as found in the hairpins of cruciforms (Furlong et al., 1989) (Table I), or the DNA is otherwise perturbed to expose the 5,6 double bond. The B-DNA-Z-DNA junctions, for instance, are hyperreactive towards OsO, oxidation although the structure of the DNA junction is not known (Johnston and Rich, 1985). It is even possible to probe the presence of Z-DNA in vivo with OsO, (Palecek et al., 1987a; 1988b). Furthermore, other unusual DNA conformations supposedly involving a combination of triple helix and single strand regions (H-DNA (Mirkin et al., 1987)) have been detected by 0.~0,-probing(Palecek et al., 1988; Vendetti et al., 1988), and specific regions of some promoters do show reactivity towards OsO, oxidation (Arcangioli and Lescure, 1986; Sverdlov et al., 1987) (cf. Table 1). Finally, Os0,-oxidation can be used to probe single base pair mismatches involving thymine or cytosine (Cotton et af., 1988). Changes in the DNA structure induced by binding of the bis-intercalating depsipeptide echinomycin (and related compounds) are also detectable by thymine Os0,-oxidation (McLean and Waring, 1988; McLean et al., 1989b). The structural changes in the DNA helix that are responsible for the increased susceptibility towards OsO, are not known at present, but helix rise, i.e., base pair/base pair distances, base tilt and base pair twist angles are parameters that would be expected to influence thymine oxidation by OsO,. 2.1.4 Potassium permanganate (KMnO,). What was said above about oxidation of DNA by oSo4 also largely holds true for potassium permanganate (KMn04), 6 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3,No. 1,1990

except that the oxidation proceeds all the way to the diol (Bayley and Jones, 1959; Sagelsdorff and Lutz, 1988). Like OsO,, KMnO, also recognizes altered DNA structures exposing the thymine 5,6 double bond. For instance, specific thymines in the middle of a DNA loop produced by the cooperative binding of lac-repressors to two operators 93 base pairs apart exhibit KMnO, hyperreactivity (Borowiec et al., 1987). Furthermore, several thymines of the RNA polymerase/promoter open complex are oxidized by KMnO, (O’Halloran et al., 1989; Jeppesen and Nielsen, 1989b; Sassa-Dwight and Gralla, 1989) probably due to a single stranded-like conformation. Finally, thymines in ordinary B-DNA are oxidized by KMnO, in the presence of intercalators and in particular bis-intercalators (Fox and Grigg, 1988; Jeppesen and Nielsen, 1988). The molecular basis of the hyperreactivity of thymines under these conditions is not known, but it is most likely connected to the helix extension and unwinding induced by intercalation as discussed for OsO, probing. Both OsO, and KMnO, must be designated as newcomers in the field of DNA probing. There is little doubt, however, that they will quickly become established probes for DNA conformations involving base pair opening of various kinds. The available evidence also suggests that identical features of the DNA are probed by both OsO, and KMnO,, so for technical reasons including safe handling (OsO, is highly toxic) KMnO, will probably be the reagent of choice, although it is a stronger and thus more indiscriminate oxidant than OsO,. Detection of the resulting purimidine diols can be done either by base catalysed DNA backbone cleavage (Friedmann and Brown, 1978; Rubin and Schmid, 1980) or by a primer extension assay (Hayes and LeClerc, 1986) because the presence of a thymine diol on the template impedes the function of DNA polymerase. It is also worth noticing that probing of RNA polymerase/promoter complexes in vivo has been reported using KMnO, (Sassa-Dwight and Gralla, 1989). 2.1.5 Aldehydes. Aldehydes, in particular formaldehyde

and glutaraldehyde, are widely used for fixation of microscopy samples. It is, therefore, not surprising that aldehydes can be and are used to crosslink protein/DNA complexes (Table 1). An ingenious technique developed by Simpson (1976) and refined by Mirzabekov and co-workers exploits the methylation of purines in protein/DNA complexes to induce depurination and concomitant unmasking in situ of the aldehyde group of deoxyribose. Proximal amino groups, e.g., of lysines, can form a Schiff base with this aldehyde and the protein/DNA crosslink thus formed is trapped by reduction with NaBH, (Simpson, 1976; Mirzabekov et al., 1978; Mirzabekov, 1980; Table I). This crosslinking procedure also results in DNA strand scission at the site of crosslinking and this site may thus be characterized by the length of the DNA fragment attached to the protein using a two-dimensional gelelectrophoretic approach. Using this technique, Mirzabekov and co-workers have mapped the positions of the histones in the nucleosome (Mirzabekov, 1980; Shick et af., 1985; Bavykin et al. 1985) and the contacts to promoter DNA of the RNA polymerase subunits



(Chenchick et al., 1981). Recently, the approach has also been applied to in vivo studies on the association of histones with actively transcribing versus inactive genes by hybridization technology (Studitsky et al., 1988). A method based on formaldehyde crosslinking and hybrid selection of the crosslinked protein/DNA complexes has been developed by Schouten (1985a) for analysing proteins associated with specific DNA sequences in vivo. A related approach involves immunoprecipitation of in vivo formaldehyde crosslinked protein/DNA complexes followed by identification of the precipitated DNA sequences by hybridization (Solomon and Varshavsky, 1985; Solomon et al., 1988). The use of aldehydes (either exogenously supplied or formed in situ) for crosslinking proteins to DNA cannot be said to have gained the status of an established technique, probably because the procedures are rather cumbersome. There is, on the other hand, a need for techniques that allow in vivo analysis of the protein parts of protein/DNA complexes, so hopefully these and related procedures (cf. Section 3.1.3) will be improved and refined in the future. Aldehydes have also been used to analyse non-B-DNA conformations. Bromo- and chloroacetaldehyde both form cyclic adducts with N1-N6 of adenines (ethnoadenine, 5 ) and N3-N4 of cytosines (Kayasuga-Mikado et al., 1980) only if these positions are not engaged in hydrogen bonding. Thus, the reactions do not take place in B-DNA. B-DNA-Z-DNA junctions are, however, very reactive and can be detected by this reaction (Kohwi-Shigematsu et al., 1987). Cruciform hairpin DNA can also be detected by reaction with bromoacetaldehyde as well as with glyoxal (Furlong et al., 1989). Indirect evidence has also been presented that B-Z junctions and the triplex H-form DNA is reactive towards glyoxal (Palecek et al., 1987b; Vojtiskova and Palecek, 1987).

5

2.2 DNA sugar phosphate backbone 2.2.1 Ethylnitrosourea (ENU, ethylation). N-Ethyl-Nnitrosourea (6) reacts (presumably via diazoethane) with the phosphates of the DNA backbone forming phosphotriesters. Two stereoisomeric triesters can be formed with the ethyl groups being in either the minor or the major groove (Fig. 3), and the relative yield of these in the ethylation of B-DNA is not known. The site of ethylation can be identified by alkaline hydrolysis due to the relative lability of the phosphotriester (Siebenlist and Gilbert, 1980).

8

H2N-C-N

,NO C ‘ H,

CH,

6

Phosphate ethylation is widely used for interference studies in order to identify phosphates that are essential for protein binding, e.g., in the studies of repressoroperator (Pabo and Sauer, 1984; Heuer and Hillen, 1988) and RNA/polymerase promoter (Siebenlist et al., 1980; Siebenlist and Gilbert, 1980) interactions (cf. Table 1). The interference of protein binding by DNAphosphate ethylation is most likely due to steric hindrance by the ethyl group combined with the removal of

Figure 4. Molecular graphics model of intercalator (reagent 1 2 ) - B - D N A complex. Notice the photoactive ligand protruding into the minor groove. In case of MPE, the acridinyl moiety is replaced by methidium and the EDTA moiety is in the minor groove.

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P.E. NIELSEN

the negative charge of the phosphate diester, but could also to a lesser extent be influenced by changes in the DNA conformation as indicated by ‘H and 3’P NMR results (Pramanik and Kan, 1987). Ethylation interference is today’s established technique of choice for studying ligand interactions with the phosphates of the DNA backbone. It is an interference principle, however, and therefore it does not directly identify contacts in the complex, but rather phosphates that carry an indispensable negative charge or which are too close to the ligand to accommodate the ethyl group. It is also quite possible that phosphates that do constitute ‘points of contact’, but which are not pertinent for binding may be ‘missed’ by the ethylation interference approach. This is of course most likely in large complexes involving a higher number of contacts (cf. Section 3.2.1). Finally, it is worth mentioning that direct footprinting experiments on ligand/DNA complexes are not feasible using ENU due to the harsh conditions required for DNA modification (50% ethanol, 50 “C) (Siebenlist and Gilbert, 1980). 2.2.2 Methidiumpropyl-EDTA/FeII (MPE). The synthetic

footprinting reagent methidiumpropyl-EDTA/FeII (MPE, 7) was developed by Dervan and co-workers (Hertzberg and Dervan, 1982) by tethering the DNA

experiments with proteins and low molecular weight drugs (Table 1). Studies of the chromatin structure in nuclei have also been performed; exploiting the finding that the internucleosomal linker DNA is preferentially cleaved by MPE (Cartwright et al., 1983; Cartwright and Elgin, 1984; 1986). The sequence neutrality of the DNA cleavage by MPE has made this reagent an attractive alternative to DNaseI for footprinting analyses although MPE is more difficult to handle technically since it cleaves DNA much less efficiently than DNaseI. The large number of different systems analysed by MPE footprinting in different laboratories (Table l), do, however, clearly demonstrate the utility of this now established, general footprinting method. 2.2.3 l,l0-Wenanthroline/CuI. The ‘nuclease’ activity of the 2: 1 1,lO-phenanthroline CuI ‘complex’ (OP,Cu) (OP= orthophenanthroline, 8) in the presence of H 2 0 2 was discovered by Sigman and co-workers (Sigman et al., 1979; Sigman, 1986). DNA cleavage is due to deoxyribose oxidation probably via primary attack at the C-I’ and, to a lesser extent, at the C-4‘ positions in the minor groove of the helix (Goyne and Sigman, 1987). The OP2Cu DNA cleavage appears to be ‘a sensitive

+

8

7

intercalating ligand methidium (alias Dimidium, the methyl analogue of ethidium) via a propyl linker to the chelator ethylenediaminetetraacetic acid (EDTA). EDTA forms a stable complex with ferrous ion (Fe2+) which in the presence of oxygen (in the form of 0, or better H,O,) induces single strand breaks in DNA (Henner et al., 1982; Bull et al., 1983). The DNA backbone scissions are due to oxidation of the deoxyriboses possibly via hydroxyl radicals (Hertzberg and Dervan, 1984). MPE is thus expected to induce DNA backbone scissions proximal to the site of intercalation and footprints produced by this reagent reflect hindrance of intercalation (cf. Fig. 4). MPE cleaves DNA with no base preference and with only little sequence dependency. The borders of the footprint are therefore easily determined a t the DNA sequence level. MPE has been widely used both by Dervan and co-workers as well as by others to perform footprinting 8 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 1,1990

probe of DNA microheterogeneity since it is highly sequence dependent without any significant base dependency (Veal and Rill, 1988). It has been suggested that the cleavage efficiency reflects the geometry of the minor groove (Sigman, 1986), but no exact structural interpretations can be made at this stage. OP,Cu footprinting has almost exclusively been used by Sigman, Spassky and Buc in the analyses of promoter conformation and RNA polymerase promoter complexes (Spassky and Sigman, 1985; Spassky, 1986; Busby et al., 1987; Amouyal and Buc, 1987; Kuwabara and Sigman, 1987; Spassky et al., 1988), and in a new interesting application, Sigman and co-workers have shown that OP,Cu footprinting is feasible on protein/ DNA complexes in polyacrylamide gels. It is, therefore, possible to combine the band-shift (gel retardation) technique (Fried and Crothers, 1981) with OP,Cu footprinting (Kuwabara and Sigman, 1987) so as to obliterate the need for pure protein preparations, since footprints of specific complexes can be obtained from (nuclear) extracts (Law et al., 1987). It is difficult at present to assess the general utility of the OP,Cu footprinting method, and it is best characterized as an emerging technique. Since in most aspects it is comparable to the established MPE method and at the same time is highly DNA sequence dependent, it will probably mostly constitute a complement to the latter. It may, however, have wider applications in probing DNA conformations.


CHEMICAL AND PHOTOCHEMICAL PROBING O F DNA COMPLEXES

2.2.4 EDTA/FeII (‘hydroxyl radical’). Tullius and co-

workers have exploited the DNA cleaving properties of the ‘Fenton-like’ reaction (Fenton, 1984) of the EDTA/ FeII complex in the presence of HzOZfor footprinting (Tullius and Dombroski, 1985; 1986; Tullius, 1987; Tullius et a/., 1987). The Fenton reaction involves decomposition of H202in acidic medium with concomitant oxidation of Fe2+ and intermediate formation of hydroxyl radicals (Scheme 3, the Haber-Weiss cycle (Haber and Weiss, 1934)). It is, therefore, postulated that DNA cleavage by EDTA/FeII is due to oxidation of deoxyribose of the DNA backbone by hydroxyl radical attack and that footprints reflect close ligand/DNA backbone contacts (Tullius and Dombroski, 1986; Tullius, 1987). This interpretation is supported by the footprinting results obtained with h-repressor- and croprotein/O, 1 operator complexes showing a correspondence between the deoxyriboses protected from EDTA/Fel I cleavage by the proteins (Tullius and Dombroski, 1986) and the protein/DNA contacts determined by ethylation interference and x-ray crystallographic techniques (Pabo and Sauer, 1984; Jordan and Pabo, 1988). ‘Hydroxyl radical’ footprinting is thus used to determine ligand/DNA backbone (deoxyribose) contacts (Table 1). Fe2’

+ H 2 0 2 + H f - + F e 3 + + HO-+ H 2 0

HO. t HzOZ -tHOz’+ H2O HOz*+Fe3+ -)02+ Fe2++ H + Scheme 3.

Tullius and co-workers have also shown that EDTA/ FeII DNA cleavage, which is base-independent, can be used to study DNA conformations. In particular, bent DNA from Trypanosome kinetoplasts exhibits a striking 10 base-pair modulation of the relative cleavage, which was interpreted in terms of the width of the minor groove being widest on the outside and narrowing towards the inside of the DNA bend (Burkhoff and Tullius, 1987; 1988). This interpretation was recently disputed by Haran and Crothers (1989), however. Finally, cruciform DNA structures like the ‘Holliday’ recombination intermediate can be analysed by ‘hydroxyl radical’ footprinting (Churchill et a/., 1988). It is probably fair to say that the EDTA/FeII footprinting method has just joined the established techniques. It is being successfully employed in many laboratories and with ligand/DNA complexes of vastly different character (Table 1).

2.3 Other chemical probes A variety of other reagents that modify DNA have found applications as DNA footprinting probes. Barton and co-workers have prepared and examined a large number of chiral tris-phenanthroline transition metal (in particular Ru) complexes (9) that bind to and cleave DNA (Barton, 1986). Since DNA is chiral in itself, viz. right-handed A- and B-DNA as opposed to left-handed Z-DNA (Zimmermann, 1982; Rich et al., 1984) some of the chiral metal complexes discriminate between the various DNA forms and conformations as regards binding and cleavage and may thus be used as probes for DNA conformations (Barton, 1986; Mei and 0Heyden & Son Limited, 1990

2.

2.

Barton, 1986; 1988). In one case, evidence for an hydrolytic mechanism was presented (Basile et a/., 1987). This is interesting since the DNA cleavage products are substrates for DNA metabolic enzymes, e.g., DNA ligase. Tetrachloropalladinate (PdCl:-) causes selective depurination at adenines in DNA (Iversen and Dervan, 1987) and has been tried as a probe for echinomycin DNA interaction without marked success (McLean and Waring, 1988). Hydroxylamine can be used as a cytosine specific reagent in DNA sequencing methodology (Rubin and Schmid, 1980) and has also found application as a probe for ‘single stranded like’ DNA regions such as B-Z-DNA junctions (Johnston and Rich, 1985) or even single base pair mismatches involving pyrimidines (Cotton et a/., 1988). A very interesting, but very specialized approach, was taken by Martin and co-workers (Martin and Holmes, 1983; Murray and Martin, 1988) who incorporated 1251 into the dye Hoechst 33258 and obtained impressive footprints by incubation of this with DNA for weeks in the frozen state. The radiation produces DNA strand scissions at the binding sites for the drug. Using a somewhat related approach, Dervan and co-workers have synthesized a large number of distamycin and netropsin analogues to which an EDTA ligand is attached. In this way the binding sites of these reagents, which are in the minor groove of DNA within AT rich regions (Fig. 5), can be determined through the DNA cleaving activity of the EDTA/FeII moiety (Schultz and Dervan, 1983, 1984; Dervan, 1986; Youngquist and Dervan, 1987; Griffin and Dervan, 1986). Due to their applications in cancer therapy, cis-diamminedichloroplatinum I1 (cis-DDP) and other platinum complexes have been known for a long time to produce both DNA interstrand crosslinks as well as DNA protein crosslinks (presumably between guanosine residues in the DNA and lysines in the proteins) (see Sherman and Lippard, 1987, for a recent review). Such platinum complexes may also have general applications as analytical tools for crosslinking protein to DNA (or RNA) (Filipski et al., 1983; Olinski ef a/., 1987) and they are particularly interesting since the crosslinking may be reversed by treatment with mercaptans. Finally, other anticancer agents o r carcinogens which form DNA adducts or cleave DNA may be used as probes. Neocarzinostatin (Ross et a)., 1979; Zahn and Blattner, 1985; Koepsel et a/., 1986) is an example of such a compound, and nitrogen mustards have also been used (see Section 4). JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 1.1990 9

P. E. NIELSEN

phosphonates, thiophosphates or dithiophosphates, and deaza analogues of the bases can also be employed (McLean et al., 1989b). The possibilities of designing DNA substrates for answering questions about the significance of specific ligand/DNA interactions seem virtually unlimited.

3. PHOTOCHEMICAL PROBES 3.1

Attack on DNA bases

Psoralens. Psoralens (10) are naturally occurring tricyclic aromatic compounds containing two photochemically active double bonds (the 3,4 and 4'3' bonds), 3.1.1

Psoralen

10

Figure 5. Molecular graphics model of distamycin bound in the minor groove of a 6-DNA helix.

Apart from the reagents mentioned above, a significant number of natural compounds have been shown to possess DNA-cleaving activity. Since these compounds are often cytotoxic and thus are of pharmaceutical interest, their DNA cleaving activity is being studied in its own right (e.g., Sugiura et al., 1984; Scannell et al., 1988). Bleomycin-an antibiotic that binds Fe2+-is probably the most extensively studied DNA cleaving reagent (Hecht, 1986; Stubbe and Kozarich, 1987), and parallels from results with bleomycin are often drawn when the DNA cleavage mechanism of the synthetic reagents, such as OP,CuI or MPE/FeII is studied. Carrying the base modification interference approach a stage further, one can make DNA molecules that are partially depurinated or depyrimidated (DNA missing guanines can be prepared via methylation and DNA missing guanines and adenines by acid treatment, for instance) and use this DNA in a gel retardation/ interference assay. Thus, it is possible to study whether the presence of specific bases is required for proper protein recognition (Brunelle and Schleif, 1987). Analogously, DNA pretreated with EDTA/FeII can be used to study the effects of missing nucleosides (Hayes and Tullius, 1989). The fast progress in the development of procedures for chemical and enzymatic synthesis of oligonucleotides containing modified bases or phosphates opens new possibilities for studying the requirements in terms of DNA structure for proper ligand/DNA recognition. For instance, the influence of the phosphates may be analysed by using oligonucleotides containing methyl10 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 1,1990

which can engage in photocycloaddition to, e.g., the 5,6 double bonds of thymines. Psoralens, furthermore, intercalate into double stranded DNA, and in the intercalation complex the psoralen 3,4 and 4'3' double bonds are almost perfectly aligned for cycloaddition with the thymine 5,6 double bonds. Psoralens thus photoreact quite efficiently with thymines in DNA (4 lo-*) whereas they are relatively photostable in the absence of DNA (+= depending on the psoralen derivative (Hearst, 1981)). The photochemical yields of psoralenDNA photoadducts can, therefore, be surprisingly high (yields as high as 50% or more are often possible). At DNA sequences containing adjacent thymines in opposite strands, i.e., TA and in particular 5'-TA sequences (Gamper ef al., 1984; Zhen et al., 1986; Sage and Moustacchi, 1987) one psoralen molecule can photoreact with both thymines and thus create a DNA interstrand crosslink (Fig. 6) (see Ben-Hur and Song, 1984; Cimino et al., 1985 for reviews on psoralen photochemistry and photobiology). Psoralens and in particular 4,5',8-trimethylpsoralen were the first photochemical probes to be used extensively for studying DNA secondary structure and DNA/ protein complexes, particularly in chromatin. These studies, which were pioneered by Hearst and co-workers (Hanson et al., 1976) and Cech and co-workers (Cech and Pardue, 1976), exploited the capacity of psoralens to photocrosslink the DNA strands of the double helix. The DNA psoralen crosslinking sites can be analysed by electron microscopy under conditions where the DNA strands are separated except at the crosslinked sites. Recently, Koller, Sogo and co-workers have refined this technique and performed very impressive studies on chromatin during transcription and replication (Thoma and Sogo, 1988; Sogo et al., 1989) (Table 1). For instance, they have shown by crosslinking the nascent RNA chain to the DNA template in vivo that the nucleosomal structure in SV40 is preserved during transcription. Psoralen photofootprinting is based on the

-


CHEMICAL A N D PHOTOCHEMICAL PROBING OF DNA COMPLEXES

approaches (Ostrander et al., 1988b; Zhen, 1989; Kochel and Sinden, 1989). Intercalative binding of ligands to DNA induces unwinding of the DNA helix (typically 1 &30") (see Wakelin, 1986; Neidle and Abraham, 1984; and Waring, 1981 for reviews on intercalation). It is, therefore, not surprising that intercalators including psoralens bind with higher affinity to supercoiled DNA which is already underwound. Sinden and co-workers have exploited this property of the psoralens and devised a protocol by which the torsional tension of DNA in vivo can be analysed (Sinden et al., 1980; Kochel and Sinden, 1989). This technique can also be used to monitor Z-DNA formation in vivo if a 5'-TA sequence is present in the Z-forming region or a 5'-AT sequence is present at the B-Z-junction: 5'-TA sequences are the most efficient psoralen photocrosslinking sites in B-DNA (Gamper et al., 1984; Zhen et al., 1986; Sage and Moustacchi, 1987) whereas Z-DNA cannot be crosslinked efficiently by psoralen (Sinden and Kochel, 1987). 5'-AT sequences, on the other hand, are inefficiently crosslinked in B-DNA but seem to exhibit a hundred fold increase in reactivity at B-Z-junctions (Kochel and Sinden, 1989). By combining the principle of orienting linear polyelectrolytes like the chromatin fiber in an electric field, with flash irradiation with polarized light, Crothers and co-workers have used the psoralen-DNA photoreaction to study the orientation of the internucleosomal linker DNA in the 30 nm chromatin fiber (Mitra et al., 1984; Sen et al., 1986). This photochemical linear dichroism technique takes advantage of the fact that the intercalated psoralen is oriented parallel to the DNA bases, and that only light polarized in the plane of the psoralen molecule will be absorbed and thus give rise to photoaddition. The orientation of the psoralen and thus the DNA relative to the chromatin fiber can, therefore, be calculated from the degree of photoreaction oc'curring with light polarized parallel versus perpendicular to the electric field. The DNA photoreactivity of psoralens has also been exploited to design reagents that can crosslink DNA to protein, either by tethering the psoralen to a thiol which in turn can react with conventional protein crosslinking reagents (Welsh and Cantor, 1984) or by linking a psoralen to an azido benzoyl moiety (Elsner et al., 1985). In the latter case a cleavable disulfide linker was employed. These photocrosslinking reagents have not gained much attention, however, probably due to their relatively poor efficiency ( 6 1 % of the added reagent gives rise to protein/DNA crosslinks in chromatin (Elsner et al., 1985)). It is evident from the above presentation that psoralens are well established and extremely versatile photochemical probes for studying protein/DNA interactions in general, and chromatin structure in particular, as well as DNA conformation both in v i m and especially in vivo. Several recent protocols have, furthermore, made it possible to perform these analyses at the DNA sequence level.

-

Figure 6. Schematic (above) and molecular graphics (below) model of a psoralen-thymine DNA interstrand crosslink (yellow). The structure was constructed without a DNA bend (Zhen et a/., 1 988a).

contention that only DNA regions not complexed with protein are efficiently crosslinked by psoralens. This contention is supported by the observation that the internucleosomal linker DNA in chromatin is preferentially crosslinked (Cech and Pardue, 1977; Wiesehahn et al., 1977). Due to limitations of resolution by electron microscopy, the sites of psoralen crosslinking can only be located within 10&200 base pairs using this technique. Several enzymatic protocols have, however, been developed recently allowing analysis of psoralen adduct sites at the nucleotide level (Zhen et al., 1986; Sage and Moustacchi, 1987; Ostrander et al., 1988a,b), and this principle has added a new dimension to psoralen photofootprinting (Zhen et al., 1988b; Ostrander et al., 1988a; Kochel and Sinden, 1989). In particular, it has been shown that sequence specific binding of proteins to DNA occludes psoralen photobinding (Zhen et al., 1988b). Psoralens are easily taken up by cells and the psoralen photofootprinting technique is, therefore, applicable to in vivo studies both using the electron microscopy (e.g. Cech and Karrer, 1980) as well as the enzymatic


3.1.2 Acridines. Photoactive acridines have been prepared either by synthesis of azidoacridine derivatives (e.g., 11) (Mueller et al., 1981; Kopacz et al., 1985; Jeppesen et al., 1988a) or by tethering a photoactive JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 1,1990 11

P.E. NIELSEN NH2

cH30mJ$@ N3

11

12

ligand to 9-aminoacridine via a polymethylene linker (e.g., 12) (Nielsen, 1982; Nielsen et a/., 1983, 1988b,c; Buchardt et a/., 1987). These photoactive acridine derivatives photoreact with DNA producing adducts and/or single strand scission (eventually after treatment with alkali) and some of them (compounds 11, 12) have shown potential as photofootprinting reagents (Table 1). The DNA photocleavage by the diazo-reagent 12 is almost sequence neutral (like MPE/FeII) and photofootprinting of several DNA binding drugs (distamycin, DAPI, Hoechst and echinomycin) has been performed with this probe (Jeppesen and Nielsen, 1989a). Protein/DNA photocrosslinking reagents consisting of an azidoacridine moiety linked (e.g., via a cleavable disulfide bridge) to an azidobenzoyl moiety have also been described, but these reagents exhibit rather low crosslinking efficiency ( * 3% in terms of added reagent when crosslinking histones to DNA in chromatin (Nielsen et al., 1984)). Finally, diacridines linked to an azidobenzoyl ligand have been prepared (e.g., 13) (Nielsen, 1982, 1985). These reagents were designed as photolabeling reagents

for DNA associated proteins. The diacridine ensures high affinity for DNA by bis-intercalation while the azidobenzoyl ligand photoreacts with nearby proteins (or the DNA). The technique is probably most correctly termed affinity mediated photolabeling and has been used for studies of histone DNA interactions in chromatin, and can also be applied to in vivo situations (Nielsen, 1982; 1985; Nielsen et al., 1985). In an effort to improve the possibilities of using both photocrosslinking and photoaffinity reagents for in vivo experiments, biotinyl containing analogues of compound 13 and an azidoacridine-azidobenzoyl reagent have been synthesized (Nielsen and Buchardt, unpublished). It is, however, too early to evaluate the prospects of this approach. 12 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 1,1990

3.1.3 Ultraviolet radiation (UVB). Short wavelength ultraviolet radiation (UVB, 250 n m b h d 3 0 0 nm) has been used both as a photocrosslinking and a photofootprinting ‘reagent’ to study protein/DNA interactions. It has long been known that UVB radiation causes covalent protein-DNA crosslinks in chromatin, and it has been used as such as a ‘zero-length’ crosslinking reagent (e.g., Celis et al., 1976; Martinson et a/., 1976; Hillel and Wu, 1978). It is, however, only recently that the potential of this technique, powerful in principle, is becoming exploited. A protocol for analysing proteins associated with a specific region of the genome in vivo has been presented using nucleic acid hybridization purification procedures and radiochemical labeling (Schouten, 1985b). Asking more or less the reverse question, Gilmour and Lis devised a technique in which in vivo photocrosslinked protein/DNA complexes are immunoprecipitated with antibodies against specific proteins (e.g., topoisomerase or RNA polymerase). Following protease treatment, the DNA of this precipitate is subjected to Southern blot analysis using DNA hybridization probes for the gene of interest (e.g., Gilmour and Lis, 1984; 1985; Gilmour et al., 1986). Finally, pulsed laser (ns pulse) and flash irradiation @s pulse) photocrosslinking are attracting increased attention for time resolution studies of protein/DNA interactions (Park et al., 1980; 1982a,b; Wu et al., 1983; Singer and Wu, 1987; Hockensmith et al., 1986; 1987). It is generally assumed that UVB-photocrosslinking is rather indiscriminate in terms of yields for different proteins and that successful crosslinking is mainly determined by protein/DNA proximity. Unfortunately, the photochemistry responsible for protein/DNA crosslinking is only poorly understood (see Shetlar, 1980; Cadet et af., 1985 for reviews). The only photoreaction that has been analysed in detail, furthermore, indicates that-not too surprisingly for photochemists-protein/ DNA photocrosslinking reactions are very specific on both the protein as well as on the DNA level. Saito and co-workers have found that thymines in DNA photoreact with primary amines including lysines in proteins (Saito and Matsuura, 1985). This results in crosslinking of the amine to the DNA and subsequently in a thermal reaction ‘transfer’ of the thymine to the amine (protein) accompanied by DNA strand scission (Scheme 4). Recent results of photocrosslinking of RNA polymerase to promoter DNA (Jeppesen et al., 1988b) also demonstrate the extreme specificity of at least some protein/ DNA photoreactions. The crosslinking takes place to thymine in position + 1 of the template DNA strand. A very interesting development of protein/DNA photocrosslinking using high intensity ultra-short (picosecond) laser irradiation was recently reported (Angelov

A9

H3

+RNH2

H

k Scheme 4


1990


et al., 1988). The high light intensity results in double excitation (two photons) photochemistry which is predominantly free radical chemistry. Thus in this case more efficient and less discriminate protein/DNA photocrosslinking is expected. The very specialized equipment required for the irradiation naturally limits the general applicability of the double excitation principle. The Dhotochemistrv of the bases in the DNA helix can also be'exploited for photofootprinting. It is well known that thymine dimers (15) and pyrimidine-6,4-pyrimidone adducts (14) are formed upon irradiation of DNA (Hruska et a/., 1975; Rycyna and Alderfeld, 1985; Cadet ef al., 1985 (review)). Several protocols for determination

(plus 6,4 adduct) formation in the nucleosome core (Gale et al., 1987; Gale and Smerdon, 1988). Dimer (plus 4,6 adduct) formation is most efficient on the outside of the DNA loop.

3*2 DNA '"gar phosphate backbone 3.2.1 Uranyl salts. The uranyl(V1) ion, UO,Z+,which is a

powerful oxidant in the exited state (Burrows and Kemp, 1974), was recently shown to photocleave DNA (Nielsen et al., 1988a). The photocleavage is almost sequence neutral and can be accomplished with long wavelength ultraviolet radiation (300G hG420 nm) with a reasonable quantum yield (4 for cleavage) (Nielsen ef al., unpublished). The photocleavage is believed to involve oxidation of deoxyriboses proximal to uranyl ions complexed to phosphates of the DNA backbone (Jeppesen and Nielsen, 1989b). In the case of the A-repressor/O,l operator DNA complex, the uranyl photofootprint reflects protein DNA backbone contacts (Nielsen ef al., 1988a; Jeppesen and Nielsen, 1989b), since the protected sites closely resemble those identified by phosphate ethylation interference studies. Uranyl photofootprinting is, therefore, likely to identify phosphates that cannot complex with the uranyl probe due to their engagement in protein contacts. Uranyl photoprobing can also be used to study DNA conformations as exemplified by the RNA polymerase/ promoter open complex where hypersensitive sites are found in the - 9 to + 3 region of the promoter (Jeppesen and Nielsen, 1989b), and in the case of bent kinetoplast DNA which exhibits a modulated uranyl cleavage pattern analogous, but not identical, to that observed with EDTA/FeII (Jeppesen and Nielsen, in preparation). The uranyl photofootprinting method is still in the exploratory state of an emerging technique, but the results so far look very promising. Uranyl may be the only reagent to date that can probe ligandiphosphate contacts by protection and in this way complements ethylation interference studies, which are more difficult to perform. A direct comparison between uranyl cleavage protection and ethylation interference is not yet available, but results obtained with RNA polymerase/ operator complexes indicate that uranyl footprinting identifies more phosphate contacts than ethylation interference (Jeppesen and Nielsen, I989b). This probably means that only some of the protein/phosphate contacts present in these complexes (the ones identified by ethylation interference) are indispensable for complex formation.

-

14

15

of the distribution of both thymine dimers and 6,4adducts in DNA fragments have been published (RoyerPokora et af., 1981; Tullius and Lippard, 1981; Becker and Wang, 1984; Doetsch et al., 1985), and the formation of these adducts has recently been exploited to study protein/DNA interactions. The protocol developed for UVB photofootprinting by Becker and coworkers is based on selective reduction by NaBH, of the 4-carbonyl group of pyrimidines with saturated 5,6bond, such as thymine dimers and 6,4-adducts. The technique is purely empirical and relies on differences in yields of the photochemical reactions of the DNA bases in the absence or presence of proteins bound to the DNA, and it can be used both in vitro with radiolabeled DNA fragments or in vivo by genomic sequencing protocols (Becker and Wang, 1984; Selleck and Majors, 1987a,b; Wang and Becker, 1988; Becker et al., 1988; Selleck and Majors, 1988). Recently, Axelrod and Majors (1989) have described a photofootprinting protocol using Taq polymerase for detection of the photoproducts. This method probably also detects mainly thymine dimers and 6,4 adducts and the authors state that it is faster and more reproducible than the chemical method. The formation of thymine dimers and pyrimidine4,6-pyrimidone adducts in DNA require the proper alignment of the 5,6 double bond in the two reacting thymines and of the $6 double bond of one pyrimidine with the 4-C = 0 bond of another pyrimidine, respectively. This requirement is not met in B-DNA. It should, therefore, be possible to use these photoreactions as sensitive probes for disparate DNA conformations. Except for the old observation that thymine dimers are formed 50% more efficiently in single-stranded DNA as compared to double-stranded DNA (Hosszu and Rahn, 1967) this principle does not seem to have been exploited. It may, however, be the explanation for the striking 10.3 base pair modulation of thymine dimer


3.3 Other photochemical probes Some tris-phenanthroline transition metal complexes (Co and Rh in particular) cleave the DNA backbone photochemically (Barton, 1986; Fleisher et al., 1986), and as discussed in Section 2.3 these may also be used to detect non-B-DNA conformations in vitro as well as in vivo (Chapnick et al., 1988). The cleavage mechanismdepending on the particular complex-is in most cases believed to be oxidation of the bases in the DNA helix either via singlet oxygen or via electron transfer. Organic dyes such as methylene blue and eosin that via JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 1.1990

13

P. E. NIELSEN

triplet/triplet energy transfer produce singlet oxygen upon irradiation in the presence of ground state oxygen (which is a triplet) have also been used as photofootprinting reagents in two cases (Fox and Waring, 1986; Hogan et ul., 1987). The DNA cleavage efficiency of singlet oxygen, which is due to oxidation of guanosine residues (Friedman and Brown, 1978), and which requires alkaline hydrolysis is rather low, however. Yielding and co-workers have prepared derivatives of ethidium in which one or both aminogroups were converted to azides, and used these azidoethidium reagents for photoaffinity labeling experiments on DNA and chromatin (Yielding, 1989; Coffman et al., 1982). Two photoactive deoxyuridine derivatives for incorporation into DNA with the aim of increasing the DNA photoreactivity for DNA/protein photocrosslinking have been prepared. 5-Bromo-2‘-deoxyuridine (BUdR) is taken up by cells and incorporated into DNA in place of thymidine, and the DNA from such cells has increased photoreactivity (Lin and Riggs, 1974). This is attributed to the 5-bromouracil which upon photolysis undergoes homolytic cleavage producing a uracil and a bromo radical (Dietz et al., 1987). DNA containing BUdR can be used both for photofootprinting and photocrosslinking experiments (Lin and Riggs, 1974; Ogata and Gilbert, 1977; Simpson, 1979a,b) since the uracil radical can either abstract a hydrogen from the deoxyribose of the DNA backbone, giving rise to a base labile site, or form a crosslink to a nearby protein. Inspired by these results, the much more photoactive 5-azido-2‘-deoxyuridine 5‘-triphosphate has been synthesized. This reagent is a substrate for DNA polymerase substituting for dTTP and can, therefore, be incorporated into DNA by primer extension or nick translation procedures (Evans and Haley, 1987). Apart from the reagents considered in this review, a large number of DNA ‘photonucleases’ which have not been used for photofootprinting have been described recently. These include such divergent reagents as acridine orange (Freifelder et d.,1961), porphyrins (Ward et ul., 1986; Bromley et al., 1986; Fiel, 1989), nitrobenzamides tethered to 9-aminoacridine (Buchardt et al., 1987; Nielsen et al., 1988b), diazopyrenium cations (Blacker et al., 1986), arylthioronium salts (Buchardt et al., 1989), bleomycin-Co (Subramanian and Meares, 1985, 1986) (used by McLean et al. (1989a) for photofootprinting of itself), promazines (Decuyper et a/., 1986) and aflatoxins (Stark et al., 1988).

4. OLIGONUCLEOTIDE PROBES I t is at the edge of the scope of this review, but the large number of oligonucleotide-based probes deserves a comment. These were made primarily in order to target a chemical or photochemical reagent to a specific RNA or DNA sequence. The probe would be attached to an oligonucleotide, which would hybridize to a complementary RNA or DNA sequence (or as recognized recently by triplex helix formation) and so direct the reagent to this sequence. Sequence specific probes to be used as molecular tools (‘synthetic restriction nucleases’ and ‘alkyl (methyl)ases’), or in a more distant, future 14 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 1.1990

gene targeted therapeutics may be developed by this strategy. The chemical and photochemical ligands so far attached to oligonucleotides include many of the ones already described as probes in their own right, namely EDTA/FeII (Boutorin et al., 1984; Dreyer and Dervan, 1985; Moser and Dervan, 1987; Strobel et al., 1988), proflavine (Praseuth et al., 1988a), azidoproflavin (Praseuth et al., 1988b), porphyrin (Le Doan et al., 1987), phenanthroline/CuI (Chi-hong and Sigman, 1986; Chen and Sigman, 1988)- nitrogen mustards (Knorre and Vlassov, 1985), methylthioether (Iverson and Dervan, 1988) and psoralens (Lee et al., 1988). A recent review on this subject is found in Stein and Cohen (1 988). In parallel approaches using chemical or photochemical probes attached to oligonucleotide primers for RNA polymerase, Grachev et al. (1989) and Stackhouse and Meares (1988, 1989) have very elegantly probed the contacts between the growing nascent RNA chain and the E. coli RNA polymerase. Similar experiments with yeast RNA polymerase have also been performed (Riva et ul., 1987).

5. ENZYMATIC PROBES Although this review is concerned with chemical and photochemical probing, it would be incomplete without a brief mention of enzymatic probes since these are still the most widely used ones for DNA footprinting. DNaseI is the enzyme first used in footprinting experiments (Galas and Schmitz, 1978) and is still the enzyme of choice for standard footprinting analyses. Other endonucleases such as DNaseII (Low et al., 1984) or micrococcal nuclease exhibit different sequence preferences and cleavage mechanism (Drew, 1984) and thus have complementary applications. Micrococcal nuclease is, furthermore, widely used to study chromatin nucleosoma1 structure since this enzyme preferentially cleaves the internucleosomal linker DNA of chromatin (Kornberg, 1977). Determination of the boundaries of protein binding sites on DNA is often performed by protocols employing exonuclease 111 (Wu, 1984; 1985), and this enzyme (Tullius and Lippard, 1981) plus other exonucleases: Ba131 (Zhen ct al., 1985; 1987) h-exonuclease (Ostrander et al., 1988a) or the exonuclease activity of T,-DNA polymerase (Sage and Moustacchi, 1987) can be used for determining the sites of drug-DNA adducts, e.g., psoralen- and cis-Pt adducts as well as thymine dimers and pyrimidine-4,6-pyrimidone adducts.

6.

PROBE/COMPLEX INTERFERENCE

Employment of probes always involves the risk of perturbing the complex under study. As discussed in Sections 2.1.1 and 2.1.2 this can be a consequence of


CHEMICAL A N D PHOTOCHEMICAL PROBING OF D N A COMPLEXES

reactions between the probe and the ligand (protein) of the complex. Since such reactions can rarely be avoided one has to take them into consideration when interpreting the results. The introduction of DNA intercalating-and other DNA binding-probes poses a special problem since DNA binding in general and intercalation in particular may perturb the complex. Intercalation is accompanied by significant changes in DNA helix parameters in terms of base-pair twist angles (unwinding) and interbase-pair distances (helix extension) (see Waring, 1981 and Neidle & Abraham, 1984, for reviews on intercalation). Indeed, it has been found that high concentrations of ethidium bromide cause weakening of histone/DNA interactions (Stratling and Seidel, 1976) and dissociation of HMG proteins (Schroter et al., 1985) in chromatin. These effects occur, however, at rather high drug/DNA ratios ( r = 0.1). Evidence by DNaseI footprinting has also been presented that the bis-intercalating depsipeptide echinomycin, the minor groove binder distamycin, and other DNA binding drugs change slightly the position of the histone core on the DNA in nucleosome core particles (Low et al., 1986; Portugal and Waring, 1987b). Distamycin has also been shown to inhibit the binding of the transcription factor OFT- 1 to its recognition sequence (Broggini et ul., 1989). DNA binding ligands thus do perturb protein/DNA complexes. On the other hand, overwhelming evidence-apparent from the data collected in Table I reporting footprinting by intercalators such as MPE, psoralens and acridines-suggests that proteins bound to DNA occlude intercalation. This is not surprising since DNA binding constants for intercalators typically are lo5M - 1 whereas sequence specific binding of, e.g., h-repressor to the recognition sequence 0,1 is -lo* times stronger ( K , = IOI3 M - ’ (Riggs ef al., 1970)). This difference in binding constants may of course be counterbalanced by a correspondingly higher concentration of the intercalator. In fact, Riggs e f al. (1970) have shown by filter binding that lo5 excess of actinomycin is capable of inhibiting binding of hrepressor to the 0,l operator and we have found by DNaseI footprinting (Nielsen et al., unpublished) that intercalators including ethidium bromide inhibit binding of h-repressor to 0,l DNA at intercalator/DNA(P) ratios exceeding 0.3 which in our experiments corresponded to an intercalator/repressor ratio of 100. It is thus important when using DNA intercalation probes to keep the concentration of the probe as low as possible. When studying conformational changes in the DNA, one should, furthermore, keep in mind that the structure seen, or ‘caught’, by the probe is not necessarily a predominant one. It could easily be (and very little work has been done to address this quite important issue) that a scarcely populated conformational state is preferentially observed due to its high reactivity.

-

-

7. CONCLUDING REMARKS

It has hopefully emerged from the preceding presentation that many aspects of ligand/DNA interactions can be analysed using chemical and photochemical probing. While some procedures are now established by virtue of their successful use in many laboratories, others must be regarded as emerging due to their obvious potential but still limited applications, often only by a single laboratory. Finally, a number of probing techniques have not yet reached an application level that allows for an evaluation of their general usefulness and they are, therefore, best regarded as experimental. It is also clear from the data that the large majority of the techniques are probing the complexes in terms of the DNA. It would be extremely informative to be able to analyse what proteins are associated with a particular region of the genome at a given time. A few protein/ DNA crosslinking techniques have addressed this question, but without gaining the widespread applications that could be anticipated for such techniques. Thus more efficient and/or sensitive techniques probably have to be developed. It should also be possible, at least in principle, to develop probes and procedures for footprinting of the proteins bound to DNA in order to analyse which regions of the proteins are contacting the DNA. Finally, photochemical probing in particular has an obvious, but as yet only sparsely exploited potential for time resolution studies. As our knowledge of static ligand/DNA complexes increases, there arises an interest for studying the dynamics of such systems, asking questions as to how a protein finds its recognition sequence in a large DNA molecule, not to mention questions concerning the functioning of the transcription and replication machineries. We shall no doubt see a boost in the in vivo probing of protein/DNA complexes in the immediate future since many biological problems can be addressed in this way and the technology is already at hand. Hopefully, we shall also experience an increased interest in studies addressing the factors in terms of DNA conformational parameters, etc., that determine probe reactivity, since this knowledge will enable us to advance much more detailed structural models for the DNA complexes on the basis of chemical and photochemical probing. The inventiveness of finding new probing methods should also keep flourishing.

Acknowledgements The financial support of the NOVO Foundation (PEN is a Hallas-Msller fellow) i s gratefully acknowledged. The author also wishes to thank Else Uhrenfeldt for typing and Dr Claus Jeppesen and D r Michael Waring for critically reading the manuscript.

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3, No. 1,1990 25

Probing DNA structure with osmium tetroxide complexes in vitro.

Probing DNA triple helix structure by chemical ligation.

Analysis of DNA-protein complexes induced by chemical carcinogens.

Photochemical water oxidation and origin of nonaqueous uranyl peroxide complexes.

Single-molecule correlated chemical probing of RNA.

Chemical probing of RNA in living cells.

DNA-osmium complexes: recent developments in the operative chemical analysis of DNA epigenetic modifications.

Photochemical reactions of chlorpromazine; chemical and biochemical implications.

Protonated triplex DNA in E. coli cells as detected by chemical probing.

Physico-chemical study of the complexes of "33258 Hoechst" with DNA and nucleohistone.

Probing chemical space with alkaloid-inspired libraries.

Chemical kinetics. Probing the transition state.

Chemical cleavage of plasmid DNA by Cu(II), Ni(II) and Co(III) desferal complexes.

Photochemical DNA modifications induced by 1,2-dioxetanes.

Probing the force-induced dissociation of aptamer-protein complexes.

Probing and perturbing stem cells with chemical biology.

Probing the elastic limit of DNA bending.

Computational analysis of RNA structures with chemical probing data.

Probing for DNA methylation with a voltammetric DNA detector.

Immunofluorescence for the detection of photochemical lesions in intracellular DNA.

Modulation of the Photophysical, Photochemical, and Electrochemical Properties of Re(I) Diimine Complexes by Interligand Interactions.

anti-DNA complexes.

Probing DNA by 2-OG-dependent dioxygenase.

Probing DNA structure with psoralen in vitro.