Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes ´ ´ ˇ ´ ´ ˇ Katerina Chvalova1,*, Jana Kasparkova1,*, Nicholas Farrell2 and Viktor Brabec1 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Department of Chemistry, Virginia Commonwealth University, Richmond, USA Keywords cross-link; conformation; DNA; DNase I; footprinting; platinum complex Correspondence V Brabec, Institute of Biophysics, Academy of Sciences of the Czech Republic, ´ ´ Kralovopolska 135, CZ-61265 Brno, Czech Republic Fax: +420 51412499 Tel: +420 541517148 E-mail: brabec@ibp.cz Website: http://www.ibp.cz/labs/BNAIAD *These authors contributed equally to this work (Received 25 April 2006, revised June 2006, accepted June 2006) Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{transPtCl(NH3)2}2l-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin) These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequencedependent binding to and cutting of DNA by DNase I It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I doi:10.1111/j.1742-4658.2006.05356.x DNA interactions of small molecules are frequently crucial events underlying their biological effects so that examinations of these interactions are of great interest The specificity of these interactions varies over an enormously wide range A number of ‘footprinting’ methods have been developed for determining the sequence-specific binding of small molecules to DNA Some of these methods are based on the ability of DNA binding molecules to protect DNA from enzymatic or chemical cleavage at their binding sites The cleavage patterns of regions to which a sequence-selective agent is bound are altered in comparison to free, nonmodified regions in DNA One subclass of footprinting agents that has been developed comprises enzymes such as deoxyribonuclease I (DNase I) [1] DNase I is an endonuclease (monomeric glycoprotein of molecular mass 30.4 kDa) that specifically cleaves the O3¢–P bond of the phosphodiester backbone of the double-helical DNA substrate It binds to the minor groove of B-DNA and cleaves each strand independently [2,3] The nuclease binds asymmetrically to the minor groove of DNA contacting two phosphates on each side of the cleaved bond and two phosphates on the complementary strand across the minor groove, opposite the phosphates contacted on the 5¢ side of the cleaved bond (Fig 1A) In addition, binding of the enzyme to DNA induces a widening of the minor groove by 0.3 nm coupled with a 21° bend towards the major groove Thus, the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, in particular the minor groove width and depth, Abbreviations BBR3464, [{trans-PtCl(NH3)2}2’-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+; cisplatin, cis-diamminedichloroplatinum(II); CL, cross-link; DMS, dimethyl sulfate; DNase I, deoxyribonuclease I; FAAS, flameless atomic absorption spectrophotometry FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS 3467 ´ ´ K Chvalova et al DNase I cleavage of platinated DNA A Fig (A) Schematic representation of the contacts made by DNase I with DNA (adapted from ref [34]) The contacts are plotted on a cylindrical projection of a DNA double helix The base pairs (short vertical lines) are drawn across the minor groove The greyshaded area represents the approximate diameter of DNase I (it is not meant to accurately represent the projected threedimensional shape of the protein) The figure shows the phosphate contacts made by DNase I at a cleavage site, taken from the structure of DNase I ⁄ DNA complex [35,36] An arrow marks the DNase I cleavage site The filled spots (d) indicate the phosphate groups contacted by DNase I to cleave at the site indicated (B) Structures of cisplatin (left) and BBR3464 (right) (C) Sequences of the deoxyribonucleotide duplexes The top and bottom strands of each pair in Fig 1C are designated ’top’ and ’bottom’, respectively, throughout The bold letters in the top and bottom strands of the duplexes indicate the platinated residues B C sequence-dependent flexibility and bendability of the double helix, are very important, indirect determinants of sequence-dependent binding to and cutting of DNA by DNase I [4] Although DNase I cuts all phosphodiester bonds, the cleavage pattern of nonmodified or noninteracting B-DNA is very uneven since the geometry of the minor groove is sequence-dependent Also importantly, DNA segments displaying an A- or Z-type conformation are resistant to DNase I digestion [4,5] Steric hindrance from ligand bound to DNA is another obvious factor decreasing affinity and cleavage efficiency of DNase I In this case, suitably placed groups of the DNA-bound ligand located in the minor groove may physically impede the approach of DNase I to the phosphodiester backbone and in this way directly prevent cleavage All this information supplies a rational basis on which the results of DNase I footprinting can be interpreted DNase I footprinting has been already used to characterize the DNA binding of a number of small molecules of biological significance [1,6–15], including antitumor cis-diamminedichloroplatinum(II) (cisplatin) (Fig 1B) and its clinically ineffective trans isomer [16] The bifunctional platinum complexes, such as cisplatin and its analogues, form various types of adducts on DNA [17,18] Recently, a trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2l-transPt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464, Fig 1B) 3468 has entered clinical trials as a potential new antitumor drug, which binds to DNA in a way which is fundamentally different from that of cisplatin [19–24] The conformational alterations induced in DNA by various bifunctional adducts of BBR3464 have been probed by a number of methods Among the unique properties of BBR3464 are the capability to form long-range crosslinks (CLs), where the platinated sites in DNA are separated by several intervening base pairs [19] Hence, the footprints of BBR3464 when it binds to DNA may be larger than those of mononuclear platinum compounds, such as cisplatin BBR3464 and cisplatin allow preparation of a broad spectrum of DNA adducts which differ significantly in the extent and character of the resulting conformational alterations induced in DNA [19] To expand the database of DNase I footprints, we compared in the present work DNA duplexes containing unique and single, site-specific adducts of trinuclear platinum complex BBR3464 and mononuclear cisplatin Results DNase I cleavage of duplexes containing intrastrand CLs of BBR3464 or cisplatin The 20 bp duplexes containing the single, site-specific adduct of BBR3464 or cisplatin were prepared as FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS ´ ´ K Chvalova et al DNase I cleavage of platinated DNA described previously [20,21,25] with a radioactive label at the 5¢ end of the top or bottom strand Nonmodified and platinated duplexes were subjected to limited DNase I cleavage [20,21,25] The digestion products were treated with 0.2 m NaCN at pH 11 to remove all platinum [26] to eliminate alterations in electrophoretic mobility caused by the charged platinum moiety and then these products were resolved on sequencing gels BBR3464 forms in natural DNA 80% intrastrand CLs of different length between guanine residues [19] In the present work, we analyzed by DNase I footprinting methodology the duplexes 1,3-IAC and 1,5-IAC (Fig 1B) containing the 1,3- and 1,5-intrastrand CLs of BBR3464, respectively Platinated guanines in the top strands of these duplexes were separated by one or three intervening bases, respectively The susceptibility to DNase I is more pronounced in the platinated strand than in the complementary strand (Figs and 3) The protected cleavage sites span approximately four and nine consecutive bonds in the 1,3- and 1,5-intrastrand duplexes, respectively The DNase I footprints of both intrastrand CLs of BBR3464 not differ only in the length of the footprint In the 1,3 intrastrand adduct, protection from cleavage occurs from the 5¢-platinated guanine and thymine on its 3¢ side and the three sites on the 5¢ side (Fig 2) In addition, the markedly enhanced cleavage by DNase I of the bond between two thymidines on the 3¢ side of the adduct in the top strand is another interesting consequence of the formation of 1,3-intrastrand CL of BBR3464 In the 1,5-intrastrand adduct, the nine cleavage sites include bases both 5¢ and 3¢ to the platinated guanines (Fig 3) However, no strong enhancement of the cleavage occurs, such as that observed for the cleavage of the duplex containing 1,3-intrastrand CL of BBR3464 on its 3¢ side Hence, the patterns of cleavage by DNase I of the duplexes containing either 1,3- or 1,5-intrastrand CL of BBR3464 are different, but a strong protection of the cleavage sites in the top strand on the 5¢ side of the adduct is the common feature of both patterns A Fig DNase I cleavage of control (nonmodified) DNA duplex 1,3-IAC and the duplex 1,3-IAC containing single, site specific 1,3-intrastrand CL of BBR3464 (A) Phosphorimage of a 24% denaturing polyacrylamide gel Lanes: 1: nonmodified duplex, 5¢-end-labelled top strand; 2: platinated duplex, 5¢-end-labelled top strand; 3: nonmodified duplex, 5¢-end-labelled bottom strand; 4: platinated duplex, 5¢-end-labelled bottom strand (B) Quantitative representation of DNase I cleavage of the nonmodified and platinated duplexes The bars represent the amount of radioactivity associated with the bands The white bars represent the cleavage in the nonmodified duplex and the black bars the cleavage in the platinated duplex (C) The differential histogram; vertical scales are in units of ln[(fa) ⁄ (fc)], where fa is the fractional cleavage at any bond in the presence of the platinum drug and fc is the fractional cleavage of the same bond in the control, nonmodified duplex, given closely similar extents of overall digestion Negative values correspond to the adductprotected site and positive values represent enhanced cleavage Data shown in Figs 2B,C are compiled from quantitative analysis of three sequencing gels (including the gel shown in Fig 2A) and must be considered a set of averaged values FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS B C 3469 ´ ´ K Chvalova et al DNase I cleavage of platinated DNA A B C Fig DNase I cleavage of control (nonmodified) DNA duplex 1,5-IAC and the duplex 1,5-IAC containing single, site specific 1,5-intrastrand CL of BBR3464 See Fig for other details Cisplatin can form minor 1,3-intrastrand CLs in DNA and it is interesting to compare the results of DNase I cleavage of the duplexes containing 1,3-intrastrand CLs of trinuclear BBR3464 and mononuclear cisplatin [22] To date this type of cisplatin adduct has not been analyzed by DNase I cleavage assay The structure of this CL is known [27] so that a concrete basis for interpreting DNase I footprinting data is available The protected cleavage sites span eight or nine consecutive bonds in both the top and bottom strands, respectively, which is considerably more than the amount of protected sites in the duplex containing 1,3-intrastrand CL of BBR3464 (Fig 4) Protection from the cleavage of the sites between the platinated guanines and the six sites in the top strand on the 5¢ side of this adduct and of the sites at complementary nucleosides in the bottom strand represent major changes in the cleavage due to formation of the 1,3-intrastrand adduct of cisplatin No markedly enhanced cleavage by DNase I in the cisplatin-1,3-intrastrand CL is observed in contrast to that observed for the analogous adduct of BBR3464 Thus, DNase I footprinting data obtained for the duplexes containing the 3470 intrastrand CLs of mononuclear cisplatin and trinuclear BBR3464 are distinctly different DNase I cleavage of duplexes containing interstrand CL of cisplatin or BBR3464 BBR3464 and cisplatin also form in DNA interstrand CLs BBR3464 forms interstrand CLs with much higher frequency than cisplatin (20% vs 6%) [19] The unique properties of interstrand CLs of BBR3464 and resulting conformational alterations in DNA are considered critical consequences for its antitumor effects [21] In contrast, contradictory data have been published on the correlation of DNA interstrand cross-linking by cisplatin and its cytotoxic effects so that it is unclear whether these DNA adducts play a decisive role in the mechanism of antitumor efficiency of cisplatin [17,28] BBR3464 forms in DNA long-range interstrand CLs between guanine residues in both directions, i.e in the 3¢)3¢ and 5¢)5¢ direction [21] The orientation of the 1,4-interstrand CL (where the platinated guanine residues are separated by two base pairs) in the 3¢)3¢ or FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS ´ ´ K Chvalova et al DNase I cleavage of platinated DNA A B C Fig DNase I cleavage of control (nonmodified) DNA duplex 1,3-IAC and the duplex 1,3-IAC containing single, site specific 1,3-intrastrand CL of cisplatin See Fig for other details 5¢)5¢ direction can be explained with the aid of the sequences of duplexes 1,4-IEC-3¢)3¢ or 1,4-IEC-5¢)5¢ (for their sequences, see Fig 1B) For instance, the 1,4-GG interstrand CL oriented in the 3¢)3¢ direction is that formed in duplex 1,4-IEC-3¢)3¢ between the central G residue in the top strand and G7 in the bottom strand, whereas the same CL oriented in the opposite, 5¢)5¢ direction is that formed in duplex 1,4IEC-5¢)5¢ between the central G residue in the top strand and G14 in the bottom strand 1,4-Interstrand CLs are formed in both directions with approximately the same frequency [21] The duplexes 1,4-IEC-3¢)3¢ and 1,4-IEC-5¢)5¢ were prepared which contained the single and central, site specific 1,4-interstrand CL between guanine residues in the 3¢)3¢ and 5¢)5¢ direction, respectively The duplexes interstrand cross-linked by BBR3464 are protected from DNase I cleavage at the sites in both strands, although the protection of the cleavage sites in the bottom strand is somewhat less pronounced (Figs and 6) The protection is always observed at the sites contained in the sequences covered by the adduct Additional protection from the cleavage is observed for the sites contained in the sequences not covered by the adduct, namely for two sites on the 3¢ side of the platinated guanines in the 3¢)3¢ adduct in each strand (Fig 5) In contrast, this additional protection in the duplex 1,4-IEC in the 5¢)5¢ direction is more extensive (Fig 6) The latter additional protection is observed for two sites on the 3¢ side of the platinated guanine in each strand and for four or five sites on the 5¢ side of this adduct in the top or bottom strand, respectively Taken together the protection spans, respectively, approximately eight or 12 base pairs if the interstrand CL is formed in DNA in the 3¢)3¢ or 5¢)5¢ direction by BBR3464 No enhancement of cleavage was seen We also prepared the 1,2-IEC duplex (Fig 1B) which contained the single and central, site-specific FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS 3471 ´ ´ K Chvalova et al DNase I cleavage of platinated DNA A B C Fig DNase I cleavage of control (nonmodified) DNA duplex 1,3-IEC-3¢)3¢ and the duplex 1,3-IEC-3¢)3¢ containing single, site specific 1,3-interstrand CL of BBR3464 formed in the 3¢)3¢ direction See Fig for other details interstrand CL of cisplatin between guanine residues in the 5¢-GC ⁄ 5¢-GC sequences, i.e in the 5¢)5¢ direction and at the sites at which this CL is preferentially formed in natural DNA [26] The sites in the duplex 1,2-IEC are protected from DNase I cleavage due the formation of interstrand CL of cisplatin in both strands (Fig 7) The maximum protection is observed at the sites covered by the CL and the protection decreases gradually almost symmetrically in both directions from the CL The total protection spans about 11 cleavage sites in both strands, i.e more cleavage sites are protected than in the case of the long-range interstrand CLs of BBR3464 (Figs 5–7) The bonds protected from the cleavage by DNase I due to formation of the interstrand CL of cisplatin in the top and bottom strand are staggered only by one base in the 5¢ direction (Fig 7), but to a somewhat lesser extent The DNase I footprint of the interstrand CL of cisplatin obtained in the present work is different from that described previously [16] The reasons for this difference are unknown, but as it is mentioned below, our results 3472 are nicely consistent with the structural information available for this type of the adduct Discussion In general, binding to and cutting of DNA by DNase I is affected by the local structure of the DNA at and in the close proximity of the cleavage site, in particular the minor groove width and depth, flexibility of the double helix and propensity of DNA to bend towards the major groove [4] DNA duplexes containing the 1,3-, and 1,5-intrastrand CL of BBR3464 have been characterized [20] The marginal platinum atoms (joined by the linker) in the intrastrand CLs of BBR3464 are located in the major groove of DNA so that the observed alterations in the cutting patterns due to formation of the intrastrand CLs of BBR3464 are likely to arise through indirect (conformational) effects It has been shown that intrastrand CLs of BBR3464 not result in a stable curvature (directional bending), but rather these adducts increase flexi- FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS ´ ´ K Chvalova et al DNase I cleavage of platinated DNA A B C Fig DNase I cleavage of control (nonmodified) DNA duplex 1,5-IEC-5¢)5¢- and the duplex 1,3-IEC-5¢)5¢-containing single, sitespecific 1,3-interstrand CL of BBR3464 formed in the 5¢)5¢ direction See Fig for other details bility of the duplex [20] An increased flexibility introduced to the helix in this manner is supported by the observation that these lesions create a local conformational distortion which spans several base pairs and mainly occurs on the base pairs covered by these adducts and on their 5¢ side The results of the present work demonstrate that the bonds mainly in the top strands of the duplexes containing intrastrand CLs of BBR3464 are protected from DNase I cleavage (Figs and 3) This protection exhibits sequence preferences similar to those observed for the conformational distortions, i.e the protection also occurs at the sites covered by the adduct and at those on its 5¢ side The protected cleavage sites span more consecutive bonds in the 1,5-intrastrand CL than in the 1,3-intrastrand CL (Figs and 3) Favorable DNase I binding contacts necessary for cleavage by this endonuclease occur at the phosphate backbone and formation of the DNase I-DNA complex preceding DNA cleavage is also driven by ionic contacts between protein side chains and the phosphate backbone [30] Formation of the intrastrand CL of BBR3464 introduces on DNA an overall 6+ charge and may therefore unfavorably affect ionic contacts between positively charged DNase I side chains and the negatively charged phosphate backbone of the strand to which BBR3464 is coordinated This may explain why the susceptibility to DNase I cleavage is enhanced on the coordinated top strand On the other hand, it has also been shown that intrastrand CLs of BBR3464 induce in DNA conformational changes characteristic of the B fi A transition [29] Hence, it cannot be excluded that the protection of the top strand to which BBR3464 is coordinated may be potentiated by the capability of intrastrand CLs of BBR3464 to induce conformational alterations which have features of A-DNA [4,5] The markedly enhanced cleavage by DNase I of the bond between two thymidines on the 3¢ side of the adduct in the top strand is a unique and interesting observation for the 1,3-intrastrand CL of BBR3464 (Fig 2) It is quite plausible that the formation of the 1,3-intrastrand CL by BBR3464 induces a conformational alteration in DNA that either directly opens up the minor groove or renders it more amenable to FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS 3473 ´ ´ K Chvalova et al DNase I cleavage of platinated DNA A B C Fig DNase I cleavage of control (nonmodified) DNA duplex 1,2-IEC and the duplex 1,2-IEC containing single, site specific interstrand CL of cisplatin See Fig for other details DNase I-mediated widening in comparison with the 1,5-intrastrand CL It is interesting that enhanced cleavage by DNase I is not seen for the 1,5-intrastrand CL (Fig 3), suggesting that the structures of the two adducts differ Further studies are required to delineate these differences A 1,3-intrastrand CL can be also formed by mononuclear cisplatin [31] and the DNase I footprinting data obtained for the duplexes 1,3-IAC containing 1,3-intrastrand CL of mononuclear cisplatin or trinuclear BBR3464 are distinctly different (cf Figs 2–4) The 1,3-intrastrand CL formed by cisplatin bends the helix axis towards the major groove by 30° and locally unwinds DNA (by 19°) [27] In addition, DNA is locally denatured and flexible at the site of the adduct [27,32] The duplex is distorted from B-DNA at the sequence covered by the adduct and at several base pairs containing the base on the 5’ side of the adduct The base pairing is even lost in the base pairs containing 5¢-platinated guanine and the base between platinated guanines, and the central base is extruded from the minor groove To accommodate this lesion, the minor groove is widened, and the 5¢-guanine deoxyribose adopts an N-type conformation commonly 3474 seen for A-DNA Thus, it is evident that the conformational distortions imposed on DNA by 1,3-intrastrand CL of cisplatin are more severe than those imposed by the same CL of BBR3464 The fact that two base pairs at the level of 1,3-intrastrand adduct are denatured and that the base between two platinated guanines is extruded from the minor groove implies that conformational alteration may considerably affect the nonplatinated strand as well This implication is consistent with the observation that 1,3-intrastrand CL of cisplatin also unfavorably affects DNase I cleavage of the unplatinated strand (Fig 4), an effect not observed with BBR3464 Interestingly, no enhanced cleavage by DNase I due to the 1,3-intrastrand CL of cisplatin is observed – suggesting that the more severe conformational alterations induced by 1,3-intrastrand CL of cisplatin not allow DNase I to make more favorable contacts with phosphate backbone resulting in enhanced cleavage The 1,4-GG interstrand CLs formed by BBR3464 in the 3¢)3¢ and 5¢)5¢ directions result in a directional bending of the helix axis (21° and 15°, respectively, toward the major groove) and duplex unwinding (14° for both types of the interstrand CL) [21] In addition FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS ´ ´ K Chvalova et al to these bending and unwinding effects, the chemical probes of DNA conformation have revealed that the 1,4-interstrand CLs formed by BBR3464 create local nonsymmetrical conformational distortions [21] The patterns of distorted sites detected by chemical probes support a different character of localized conformational distortions due to the different orientation of these lesions NMR characterization of the 1,4-interstrand CL formed by BBR3464 in the 5¢)5¢ direction have revealed that within the sequence covered by the CL, the nucleosides appear to be a mixture of syn and anti conformations with Watson–Crick hydrogen bonding maintained [24] In addition, this characterization has also revealed altered conformation of the nucleosides outside the sequence covered by the CL and interactions of several groups in the minor groove with the central platinum unit of BBR3464 The latter observation supports the view that the formation of the 1,4-interstrand CLs may directly contribute to the protection from the cleavage by DNase I of the sites in the sequences covered by the adduct and observed in the present work (Figs and 6) The observed alterations in the cutting patterns due to the formation of interstrand CLs of BBR3464 also reveal protection of the cleavage sites outside the sequence covered by the adduct These alterations must arise through indirect effects, which implies that the 1,4-interstrand CLs induce delocalized conformational alterations beyond the actual binding sites of the CL This interpretation is consistent with the results of NMR analyses [24] and chemical probes [21] However, the analyses of DNase I footprints of 1,4-interstrand CLs also reveals new features of conformational alterations induced by these adducts The 5¢)5¢ CL apparently induces the larger conformational alteration in DNA extending over more nucleosides (Fig 6) Another unique feature of these CLs revealed by DNase I footprinting, hitherto not observed using other methods, is their capability to preferentially affect conformation outside the sequences covered by the adduct preferentially on its side in the direction of the platinated guanine residues in each strand, i.e in the case of the CLs formed in the 3¢)3¢ or 5¢)5¢ direction mainly nucleosides flanking the platinated guanines on their 3¢ or 5¢ sides, are affected (Figs and 6) Since the pattern of cleavage by DNase I of the interstrand CL of cisplatin obtained in the present work differs substantially from that published previously [16], we interpret our footprint of interstrand CL of cisplatin on the basis of structural data available for this type of the platinum adduct [28] Interstrand CLs of cisplatin are preferentially formed in natural DNA between guanine residues in the 5¢-GC ⁄ 5¢-GC sequence DNase I cleavage of platinated DNA [28], i.e in the 5¢)5¢ direction This adduct also induces several irregularities in DNA The guanine residues interstrand cross-linked by cisplatin are not paired with hydrogen bonds to the complementary cytosines, which are located outside the duplex and not stacked with other aromatic rings All other base residues are paired, but distortion has been found to extend over at least four base pairs at the site of the CL In addition, the double helix is locally reversed to a left-handed, Z-DNA-like form This adduct induces the helix unwinding by 76–80° relative to B-DNA and also the bending of 20–40° of the helix axis at the cross-linked site toward the minor groove It has been also demonstrated that the cis-diammineplatinum(II) bridge in the interstrand CL of cisplatin resides in the minor groove The latter finding is consistent with the view that the formation of the interstrand CL of cisplatin may directly contribute to the protection of the sites in the sequences covered by the adduct from the cleavage by DNase I and observed in the present work The observed alterations in the cutting patterns due to the formation of interstrand CL of cisplatin also reveal protection of the cleavage sites outside the platinated base pairs These alterations must arise through indirect effects, as direct DNA interaction of cisplatin in the interstrand CL is mainly localized to platinated base pairs consistent with the results of the previous analyses by other methods Interestingly, the analysis of DNase I footprint of the interstrand CL of cisplatin (Fig 7) also reveals very extensive delocalized conformational alterations induced by this type of the DNA adduct, which are even more extensive than those induced by the 1,4-interstrand CL of BBR3464 formed in the 3¢)3¢ direction and almost as extensive as those induced by the 1,4-interstrand CLs of BBR3464 formed in the 5¢)5¢ direction Another unique feature of the CLs formed by cisplatin revealed by DNase I footprinting is their capability to affect conformation outside the sequences covered by the adduct when the distortion is slightly more extensive on the 5¢ side of the platinated guanine residue in each strand Hence, the distortion induced by the interstrand CL of cisplatin is somewhat more symmetrical that those induced by the 1,4-interstrand CLs of BBR3464 In conclusion, this study illustrates how various indirect structural perturbations induced by platinum binding in the major groove translate into the minor groove, allowing their detection by DNase I probing The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS 3475 ´ ´ K Chvalova et al DNase I cleavage of platinated DNA alterations in DNA are differently recognized by DNase I Experimental procedures Starting material BBR3464 was prepared by standard methods Cisplatin was obtained from Sigma (Prague, Czech Republic) The stock solutions of platinum compounds were prepared at the concentration of · 10)4 m in 10 mm NaClO4 and stored at °C in the dark The synthetic oligodeoxynucleotides used in this work (Fig 1C) were purchased from VBC-GENOMICS (Vienna, Austria) The purity of compounds was verified by HPLC or by gel electrophoresis T4 polynucleotide kinase was purchased from New England Biolabs (Beverly, MA, USA) and DNase I from Roche (Mannheim, Germany) Acrylamide, bis(acrylamide), urea and NaCN were from Merck kgaA (Darmstadt, Germany) Dimethyl sulfate (DMS) was from Sigma (Prague, Czech Republic) [c32P]ATP was from MP Biomedicals, LLC (Irvine, CA, USA) Platinations of oligonucleotides The duplexes containing single, site-specific intrastrand CLs were prepared in the following way The single-stranded oligonucleotides (the top strands of the duplexes 1,3-IAC or 1,5-IAC shown in Fig 1C) were reacted in stoichiometric amounts with BBR3464 The platinated oligonucleotides were repurified by ion-exchange FPLC It was verified by platinum flameless atomic absorption spectrophotometry (FAAS) and by the measurements of the optical density that the modified oligonucleotides contained one molecule of the platinum complex It was also verified using DMS footprinting of platinum on DNA [33] that in the platinated top strands of all duplexes the N7 position of the two guanine residues was not accessible for reaction with DMS The platinated top strands were allowed to anneal with unplatinated complementary strands (the bottom strands of the duplexes 1,3-IAC or 1,5-IAC) in 0.1 m NaClO4 and used immediately in further experiments This annealing procedure included a rapid heating of the mixture of the complementary oligonucleotides to 60 °C followed by the incubation at 25 °C for h It was verified that under these conditions the intrastrand CLs of BBR3464 were stable for at least 24 h The duplexes containing single, site-specific interstrand CL were prepared in the slightly different way The singlestranded oligonucleotides (the top strands of the duplexes 1,2-IEC, 1,4-IEC3¢–3¢ and 1,4-IEC5¢–5¢ shown in Fig 1C) were reacted in stoichiometric amounts with the monoaqua derivative of cisplatin or BBR3464 The platinated oligonucleotides were repurified by FPLC It was verified that the modified oligonucleotides contained one molecule of the 3476 platinum complex by platinum FAAS and by measurement of the optical density Using DMS footprinting of platinum on DNA, we also verified that in the platinated top strands of all duplexes, N-7 of a single guanine (G) residue was not accessible for reaction with DMS The platinated top strands were allowed to anneal with unplatinated complementary strands (the bottom strands of the duplexes 1,2-IEC, 1,4-IEC3¢–3¢ and 1,4-IEC5¢–5¢ shown in Fig 1C) in 0.1 m NaClO4 The resulting products were separated on denaturing m urea and 12% polyacrylamide gel; the bands corresponding to interstrand cross-linked duplexes were cut off from the gel, eluted, precipitated by ethanol, and dissolved in 0.1 m NaClO4 FPLC purification and FAAS measurements were carried out using an Amersham Biosciences FPLC system with a Mono Q HR ⁄ column and with a Varian AA240Z Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite tube atomizer, respectively DNase I footprinting Double-stranded oligonucleotides, 5¢-end labeled on the top or bottom strand, were incubated at 25 °C with DNase I (0.05 units ⁄ mL) in the buffer composed of mm Tris ⁄ HCl (pH 8.0), 0.5 mm CaCl2, 7.5 mm KCl, 3.125 mm MgCl2, and lm DTT The total volume of the reaction was 100 lL Optimal enzyme dilutions were established in preliminary calibration experiments After the digestion was stopped by placing samples on dry ice followed by extraction with phenol DNA cleavage products were resolved by polyacrylamide gel electrophoresis under denaturing conditions (24% ⁄ m urea polyacrylamide gel) The autoradiograms were visualized and quantified by using the bio-imaging analyzer BAS-2500 (Fuji Photo Film Co Ltd., Tokyo, Japan) and aida image analyzer software (raytest Isotopenmessgerate GmbH, Strauben, Germany) Assignă ment of the cleavage to a particular base has been made so that it corresponds to the cleavage of the phosphodiesteric bond on the 5¢ side of that base Acknowledgements This research was supported by the Grant Agency of the Czech Republic (Grants 305 ⁄ 05 ⁄ 2030 and 204 ⁄ 03 ⁄ H016), the Grant Agency of the Ministry of Health of the Czech Republic (NR8562-4 ⁄ 2005), Ministry of Education of the CR (MSMT LC06030) and the Academy of Sciences of the Czech Republic (Grant 1QS500040581) The authors acknowledge that this work was carried out within the Institutional Research Plan AVOZ50040507 JK is the international research scholar of the Howard Hughes Medical Institute KC is supported by a doctoral fellowship from the Faculty of Sciences, Masaryk University, Brno The authors FEBS Journal 273 (2006) 3467–3478 ª 2006 The Authors Journal compilation ª 2006 FEBS ´ ´ K Chvalova et al also acknowledge that their 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