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Interaction between Cisplatin-modified DNA and the HMG Boxes of HMG 1: DNase I Footprinting and Circular Dichroism
JMB—MS 335 Cust. Ref. No. YAN 41/94
[SGML] J. Mol. Biol. (1995) 246, 243–247
COMMUNICATION
Interaction between Cisplatin-modified DNA and the HMG Boxes of HMG 1: DNase I Footprinting and Circular Dichroism D. Locker1, M. Decoville1, J. C. Maurizot1, M. E. Bianchi2 and M. Leng1* 1
Centre de Biophysique Moleculaire, CNRS Rue Charles Sadron 45071 Orleans Cedex 2 France 2
DIBIT, San Raffaele Scientific Institute, via Olgettina 58, I-20132 Milano Italy *Corresponding author
The interactions between the two boxes A and B of HMG 1 and cis-diamminedichloroplatinum(II)-modified DNA containing a single intrastrand cross-link at the d(GpG) site were studied by DNase I footprinting and circular dichroism. The DNAase I cleavage patterns of the HMG box-platinated DNA complexes are identical, the two boxes inhibiting the DNase I cutting over at least 15 and 12 nucleotide residues in the platinated strand and the complementary strand, respectively. As judged by circular dichroism, the two boxes have the same a-helical content (56%) and they induce the same conformational changes in the platinated DNA. Keywords: oligonucleotides; cisplatin; transplatin
HMG 1 proteins belong to the ‘‘high mobility group’’ class of mammalian chromatin proteins and are present in all vertebrate nuclei. Their sequence conservation, ubiquity and abundance suggest important functions for HMG 1. They appear to be involved in a large number of DNA transactions (reviewed by Travers et al., 1994; Bianchi, 1994). HMG 1 proteins have three structural domains: the N-terminal A-domain and the central B-domain are positively charged and bind to DNA, while the terminal C-domain is acidic and interacts with histones (general reviews and references therein; Bianchi et al., 1992a; Landsman & Bustin, 1993; Grosschedl et al., 1994). Although HMG 1 binds to double-stranded DNA and facilitates the circularization of DNA restriction fragments (Pil et al., 1993; Paull et al., 1993), it binds preferentially to bent DNA such as four-way junction DNA (Bianchi et al., 1992b), cisplatin-modified DNA (Pil & Lippard, 1992; Hughes et al., 1992) and DNA containing bulges (Pontiggia & Bianchi, unpublished results). The sequences of the two domains A and B of HMG 1 are homologous to each other (Figure 1) and to segments of about 70 amino acid residues called HMG box. The two boxes A and B of HMG 1 bind to the fourway junction DNA with the same affinity (Bianchi et al., 1992b), which is in favor of a similar mode of interaction with DNA. The data reported here strengthen this suggestion. We find that the two boxes A and B protect the same region of a cis-diamminedichloroplatinum(II) (cis-DDP)modified double-stranded oligonucleotide contain0022–2836/95/070243–05 $08.00/0
ing a single intrastrand cross-link at the d(GpG) site (abbreviated d(G*pG*)) from DNase I cleavage. In addition, we show by circular dichroism that the boxes A and B have the same folding and that they induce the same conformational changes in the platinated DNA. Recent studies (Hughes et al., 1992; Pil & Lippard, 1992) have shown that HMG 1 interacts with DNA modified with cis-DDP at d(GpG) and d(ApG) sites and does not interact with DNA modified with trans-DDP (trans-DDP is the stereoisomer of cis-DDP). The endonuclease DNase I has been extensively used to study the interactions between DNA and ligands (Tullius, 1989). Alterations induced in the DNA double helix by cis-DDP have been revealed by several approaches, including DNase I footprinting (general reviews and references therein; Kozelka & Chottard, 1990; Lepre & Lippard, 1990; Sip & Leng, 1993). The DNase I cleavage patterns of oligonucleotides bearing or not a single cis-DDP intrastrand cross-link d(G*pG*) are very different (Visse et al., 1991; Schwartz & Leng, 1994). The data in Figure 2 (lanes 0) confirm this observation. In the three studies (Visse et al., 1991; Schwartz & Leng, 1994; this work), the central sequences of the three oligonucleotides are d(CACTGGAACC), d(TCTTGGTTCT) and d(CCTCGGTTCT), respectively. The strong changes in the DNase I cleavage patterns occur mainly at the level of the 3' residue adjacent to the adduct and at the level of the C residue complementary to the platinated 5' G residue. The efficiency of the DNase I cutting appears to be 7 1995 Academic Press Limited
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Figure 1. Sequences of the boxes A and B of rat HMG 1 (identities and similarities for the aminoacids are indicated by asterisks (*) and dots, respectively) and of the oligonucleotides for the DNase I footprinting (1) and for the circular dichroism (2).
influenced by the distortions induced in the double helix by the adduct more than by the base sequence. The presence of HMG 1-box A does not affect the DNase I cleavage pattern of the unplatinated oligonucleotide while it does affect the cleavage pattern of the platinated oligonucleotide (Figure 2). Inspection of the gels shows that the box A inhibits the DNase I cutting over at least 15 and 12 nucleotide residues in the upper and lower strands, respectively. Interestingly, the protected footprints are similar for the non-sequence-specific HMG 1-box A and the sequence-specific Sox-5 HMG box (Connor et al., 1994). In the upper strand, the most noticeable effect is at the level of the T residue adjacent to the central d(GpG) site on its 3' side. This new DNase I-sensitive site after platination, is hardly cleaved in the complex with box A. In the lower strand, the most striking effect occurs at the C residue complementary to the platinated 5' G residue. At this site, the DNase I cutting, strongly enhanced by the platination, is inhibited by the presence of the box A. The next step was to compare the effects of box A and box B on the cleavage patterns of the platinated oligonucleotide. The DNase I cleavage patterns of the platinated oligonucleotide in the presence of the two boxes are identical (Figure 3); thus as judged by DNase I, the two boxes behave very similarly with respect to DNA. The crystallographic structure of the DNase I-DNA complex revealed that an exposed loop of the protein penetrates into the minor groove of the DNA and interacts asymmetrically with the backbone of both strands. DNase I contacts two phosphate groups on each side of the cleaved bond and two phosphate groups on the other strand across the minor groove (Suck & Oefner, 1986; Suck et al., 1988). The enhanced DNase I cleavage of the platinated DNA has been related to the distortions induced in DNA by the d(G*pG*) adduct (Visse et al., 1991; Schwartz & Leng, 1994). The adduct bends the double helix by 32–34° towards the major groove (and thus widens the minor groove) and unwinds it by 13° (Bellon et al., 1991). On the basis of the solution structure of the HMG box B determined by NMR (Weir et al., 1993; Read et al., 1993) and of several experiments (reviewed by Grosschedl et al., 1994; Falciola et al., 1994), a model has been proposed in which the extended segment of the HMG box binds into the
Figure 2. DNase I footprinting of the HMG 1 box A-DNA complex. The synthesis of the oligonucleotide and the platination at the d(GpG) site in the upper strand were done as described (Schwartz & Leng, 1994) with minor modifications. The protocol of Bianchi et al. (1992b) was followed to isolate the 2 boxes. They were further purified on a FPLC MonoS column. The double stranded oligonucleotides (0.3 mM) with a 5'-labeled upper strand (left) or lower strand (right) were subjected to limited DNase I digestion in 50 ml of buffer containing 20 mM Hepes (pH 8), 100 mM KCl, 7 mM MgCl2 , 1.5 mM CaCl2 , 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin, 2 mg of sonicated calf thymus DNA and 0.1 unit of DNase I (Boehringer). Similar results were obtained with 0.05 or 0.2 unit of DNase I. The digestions were carried out for 3 min at 20°C. The reactions were stopped by addition of 5 mM EGTA. The samples were loaded onto a denaturing 12% polyacrylamide gel. Maxam & Gilbert sequencing reactions were run in parallel (not shown). The concentrations of the box A were respectively 1.3 mM (lane 1), 6.3 mM (lane 2) and 13 mM (lane 3); no box A (lane 0). The arrows on each side of the sequences indicate the limits of the DNase I protected regions. The fragments containing the adducts migrate slower than the corresponding unplatinated fragments because the platinum residues were not removed before the electrophoresis.
enlarged minor groove of the platinated DNA (Faciola et al., 1994). The strong inhibition of the platinated DNA cutting by DNase I in the presence of the boxes supports the model. Assuming that in solution, a proficient incision by DNase I implies the same contacts between the phosphate groups and DNase I as those observed in the crystal (Fairall & Rhodes, 1992), one can deduce that the HMG box protects at least six nucleotide residues on each strand of the platinated DNA (rectangular box on the cylindrical projection of the DNA, Figure 3). It seems likely that the protected region extends more on the 5' side of the adduct but this cannot be guaranteed because of the absence of strong DNase I cleavage sites in the lower strand. On the other hand, an NMR study of the complex between SRY HMG box and DNA (King & Weiss, 1993) demonstrates the recognition of two A · T base-pairs of the DNA by
JMB—MS 335 Communication
Figure 3. DNase I footprinting of the platinated DNA in the presence of the HMG 1 box A or B. The experimental conditions were as described for Figure 2. The concentrations of the boxes were, respectively, 1.8 mM (lanes 1 and 2) and 5.3 mM (lanes 3 and 4). In the right part of the Figure is shown a schematic representation of the contacts between DNase I and the box-platinated DNA complex plotted on a cylindrical projection of the double helix. The base-pairs are drawn across the minor groove. An arrow ( 3 ) marks the DNase I cleavage site and the dots (,) mark the phosphate groups contacted by DNase I to cleave at the site. The minimal size of the protected region by HMG 1 box is indicated by a rectangle.
partial intercalation of a non-polar side-chain of the SRY box. Such an interaction could also occur in the HMG 1 box-DNA complex. We found (not shown) that a DNA containing a single trans-DDP interstrand cross-link is not recognized by the HMG 1 boxes, although the global distortion induced in DNA by the trans-DDP interstrand cross-link and the cis-DDP intrastrand cross-link present some resemblance. The trans-DDP interstrand cross-link bends the double helix towards the major groove by 26° and unwinds it by 12° (Brabec et al., 1993). It is tempting to speculate that the cis-DDP intrastrand cross-link makes easier the partial intercalation (the Pt residue is located in the major groove) while the trans-DDP interstrand cross-link prevents it (the Pt residue is located between the two strands). The boxes A and B have been further compared by circular dichroism (CD). The far ultra-violet spectra recorded down to 185 nm, are identical within experimental errors (Figure 4, 1). The secondary structure contents of the two boxes estimated by the unconstrained variable selection method (Manavalan & Johnson, 1987) are the same (a-helix, 56(2)1%; b-sheet, 6(2)2%; turn, 11(2)1%; other, 27(2)1%). We conclude that the two HMG 1 boxes A and B have the same folding. The values of the secondary structure contents are in good agreement with the CD values reported for the HMG 1 box B (Weir et al., 1993; Read et al., 1993), TCF-1 HMG box (van Houte et al., 1993) and Sox-5 HMG box (Connor et al., 1994). However, an intriguing point is that from
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Figure 4. Circular dichroism of the HMG 1 boxes A and B (1) and of the box-platinated DNA complexes (2). The spectra were recorded on a Jobin-Yvon spectrophotometer at room temperature. The samples were dissolved in 100 mM NaF, 10 mM phosphate buffer (pH 7.5), 0.5 mM DTT. The concentrations of the HMG boxes were 5 mM. In 2, (- · -) corresponds to the free platinated oligonucleotide, (- -) to the free HMG box A (or B) and (—) to the HMG box A (or B)-platinated oligonucleotide complex. Below 250 nm, the calculated spectrum (not shown) corresponding to the difference between the spectrum of the complex and the spectrum of the free platinated oligonucleotide resembles the spectrum of the free protein.
the NMR structure of the HMG 1 box B (Weir et al., 1993; Read et al., 1993) and of the HMG-D box (Jones et al., 1994) the a-helical content is about 75 %, which disagrees with the CD estimate. It has been argued that CD underestimates the a-helical content because of the presence of tyrosine (Bradley et al., 1990; Weir et al., 1993). This seems unlikely since the boxes A and B contain a different number of tyrosine residues and yet have the same CD spectra. This discrepancy between CD and NMR deserves to be explained. No differences were found in the CD spectra of the complexes between the platinated oligonucleotide and the box A or the box B; one of these experiments is shown in Figure 4, 2. The spectrum of the platinated oligonucleotide, slightly different from that of the unplatinated oligonucleotide in agreement with previous results (van Hemelrick et al., 1986), is modified by the binding of the box. Taking into account the concentrations of the protein and DNA, the contribution of the protein to the CD signal at wavelengths larger than 250 nm can be neglected. The modifications between the spectra of the free platinated DNA and of the complex (a decrease of the intensity and a small red-shift of the positive band ) reveal unambiguously a conformational change of the DNA induced by the binding of the HMG box. In this wavelength region, our CD results resemble those reported on HMG 1-DNA (Stros et al., 1994) but differ from those on Sox-5-DNA (Connor et al., 1994). It is premature to discuss further the conformational changes induced in three different DNAs by the binding of the HMG boxes. At wavelengths shorter than 250 nm, both the DNA and protein contribute to the CD signal and thus it is difficult to attribute unambiguously the observed changes to structural modifications of either one (or both) of the interacting molecules. Nevertheless, the fact that these variations are rather small allows exclusion of the
JMB—MS 335 246 possibility of a large conformational change of the protein, such as the formation of an a-helix described for the binding to DNA of GCN4 and the bZip fragment of C/EBP (O’Neil et al., 1991). In conclusion, the two boxes A and B of HMG 1, which have significantly different sequences, behave identically as judged by circular dichroism and DNase I footprinting. Work is in progress to characterize the interactions between the entire protein HMG 1 and DNA.
Acknowledgements We are indebted to Dr A. R. Rahmouni and A. Schwartz for helpful discussions. This work was supported in part by la Ligue contre le Cancer, la Fondation pour la Recherche Me´dicale, l’Association de la Recherche sur le Cancer and the E.E.C. (projects CHRX-CT92-0016, D1-92-002 and CHRX-CT94-0482).
References Bellon, S. F., Coleman, J. H. & Lippard, S. J. (1991). DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cisdiamminedichloroplatinum(II). Biochemistry, 30, 8026–8035. Bianchi, M. E. (1994). Prokaryotic HU and eukaryotic HMG 1: a kinked relationship. Mol. Microbiol. In the press. Bianchi, M. E., Beltrame, M. & Faciola, L. (1992a). The HMG box motif. In Nucleic Acids and Molecular Biology (Eckstein, F. & Lilley D. M. J., eds), vol. 6, pp. 112–128, Springer-Verlag, Berlin. Bianchi, M. E., Falciola, L., Ferrari, S. & Lilley, D. M. J. (1992b). The DNA binding site of HMG 1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins. EMBO J. 11, 1055–1063. Brabec, V., Sip, M. & Leng, M. (1993). DNA conformational change produced by site-specific interstrand crosslink of trans-diamminedichloroplatinum(II). Biochemistry, 32, 11676–11681. Bradley, E. K., Thomason, J. F., Cohen, F. E., Kosen, P. A. & Kuntz, I. D. (1990). Studies of synthetic helical peptides using circular dichroism and nuclear magnetic resonance. J. Mol. Biol. 215, 607–622. Connor, F., Cary, P. D., Read, C. M., Preston, N. S., Driscoll, P. C., Denny, P., Crane-Robinson, C. & Ashworth, A. (1994). DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5. Nucl. Acids Res. 22, 3339–3346. Fairall, L. & Rhodes, D. (1992). A new approach to the analysis of DNase I footprinting data and its application to the TFIIIA/5S DNA complex. Nucl. Acids Res. 20, 4727–4731. Falciola, L., Murchie, A. I. H., Lilley, D. M. J. & Bianchi, M. E. (1994). Mutational analysis of the DNA binding domain A of chromosomal protein HMG 1. Nucl. Acids Res. 22, 285–292. Grosschedl, R., Giese, K. & Pagel, J. (1994). HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 10, 94–100. Hughes, E. N., Engelsberg, B. N. & Billings, P. C. (1992). Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identification with high
Communication
mobility group proteins 1 and 2. J. Biol. Chem. 267, 13520–13527. Jones, D. N. M., Searles, A. M., Shaw, G. L., Churchill, M. E. A., Ner, S. S., Keeler, J., Travers, A. A. & Neuhaus, D. (1994). The solution structure and dynamics of the DNA-binding domain of HMGD from Drosophila melanogaster. Structure, 2, 609–627. King, C. Y. & Weiss, M. A. (1993). The SRY high-mobilitygroup box recognizes DNA by partial intercalation in the minor groove: a topological mechanism of sequence specificity. Proc. Nat. Acad. Sci., U.S.A. 90, 11990–11994. Kozelka, J. & Chottard, J. C. (1990). How does cisplatin alter DNA structure? A molecular mechanics study on double-stranded oligonucleotides. Biophys. Chem. 35, 165–178. Landsman, D. & Bustin, M. (1993). A signature for the HMG-1 box DNA-binding proteins. BioEssays, 15, 1–8. Lepre, C. A. & Lippard, S. J. (1990). Interaction of platinum antitumor compounds with DNA. In Nucleic Acids and Molecular Biology (Eckstein, F. & Lilley, D. M. J., eds), vol. 4, pp. 3–9, Springer-Verlag, Berlin. Manavalan, P. & Johnson, W. C. (1987). Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal. Biochem. 167, 76–85. O’Neil, K. T., Shuman, J. D., Ampe, C. & DeGrado, W. F. (1991). DNA-induced increase in the helical content of C/EBP and GCN4. Biochemistry, 30, 9030–9034. Paull, T. T., Haykinson, M. J. & Johnson, R. C. (1993). The non-specific DNA-binding and DNA-bending proteins HMG 1 and HMG 2 promote the assembly of complex nucleoprotein structures. Genes Develop. 7, 1521–1534. Pil, P. M. & Lippard, S. J. (1992). Specific binding of chromosomal protein HMG 1 to DNA damaged by the anticancer drug cisplatin. Science, 256, 234–237. Pil, P. M., Chow, C. S. & Lippard, S. J. (1993). High-mobility-group 1 protein mediates DNA bending as determined by ring closures. Proc. Nat. Acad. Sci., U.S.A. 90, 9465–9469. Read, C. M., Cary, P. D., Crane-Robinson, C., Driscoll, P. C. & Norman, D. G. (1993). Solution structure of a DNA-binding domain from HMG 1. Nucl. Acids Res. 21, 3427–3436. Schwartz, A. & Leng, M. (1994). DNase I footprinting of cisand trans-diamminedichloroplatinum(II)-modified DNA. J. Mol. Biol. 236, 969–974. Sip, M. & Leng, M. (1993). DNA, cis-platinum and intercalators: catalytic activity of the DNA double helix. In Nucleic Acids and Molecular Biology (Eckstein, F. & Lilley, D. M. J., eds), vol. 7, pp. 1–15, Springer-Verlag, Berlin. Stros, M., Stokrova, J. & Thomas, J. (1994). DNA looping by the HMG-box domains of HMG and modulation of DNA binding by the acidic C-terminal domain. Nucl. Acids Res. 22, 1044–1051. Suck, D. & Oefner, C. (1986). Crystallographic refinement ˚ resolution. J. Mol. Biol. and structure of DNAse I at 2 A 192, 605–632. Suck, D., Lahm, A. & Oefner, C. (1988). Structure refined ˚ of a nicked DNA octanucleotide complex with to 2 A DNase I. Nature (London), 332, 464–468. Travers, A. A., Ner, S. S. & Churchill, M. E. A. (1994). DNA chaperones: a solution to a persistence problem? Cell, 77, 167–169.
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Tullius, T. D. (1989). Physical studies of protein-DNA complexes by footprinting. Annu. Rev. Biophys. Chem. 18, 213–237. van Hemelrick, B., Guittet, E., Girault, J. P., Herman, F., Huynh-Dinh, T., Lallemand, J. Y., Igolen, J. & Chottard, J. C. (1986). A high melting cis-[Pt(NH3 )2 4d(GpG)5] adduct of a decanucleotide duplex. Biochem. Biophys. Res. Commun. 138, 758–763. van Houte, L., van Oers, A., van de Wetering, M., Doojies, D., Kaptein, R. & Clevers, H. (1993). The sequencespecific high mobility group 1 box of TCF-1 adopts a
247 predominantly a-helical conformation in solution. J. Biol. Chem. 268, 18083–18087. Visse, R., de Ruijter, M. Brouwer, J., Brandsma, J. A. & van de Putte, P. (1991). Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA. J. Biol. Chem. 266, 7609–7617. Weir, H. M., Kraulis, P. J., Hill, C. S., Raine, A. R. C., Laue, E. D. & Thomas, J. O. (1993). Structure of the HMG box motif in the B-domain of HMG 1. EMBO J. 12, 1311–1319.
Edited by M. Yaniv
(Received 3 October 1994; accepted 21 November 1994)
[SGML] J. Mol. Biol. (1995) 246, 243–247
COMMUNICATION
Interaction between Cisplatin-modified DNA and the HMG Boxes of HMG 1: DNase I Footprinting and Circular Dichroism D. Locker1, M. Decoville1, J. C. Maurizot1, M. E. Bianchi2 and M. Leng1* 1
Centre de Biophysique Moleculaire, CNRS Rue Charles Sadron 45071 Orleans Cedex 2 France 2
DIBIT, San Raffaele Scientific Institute, via Olgettina 58, I-20132 Milano Italy *Corresponding author
The interactions between the two boxes A and B of HMG 1 and cis-diamminedichloroplatinum(II)-modified DNA containing a single intrastrand cross-link at the d(GpG) site were studied by DNase I footprinting and circular dichroism. The DNAase I cleavage patterns of the HMG box-platinated DNA complexes are identical, the two boxes inhibiting the DNase I cutting over at least 15 and 12 nucleotide residues in the platinated strand and the complementary strand, respectively. As judged by circular dichroism, the two boxes have the same a-helical content (56%) and they induce the same conformational changes in the platinated DNA. Keywords: oligonucleotides; cisplatin; transplatin
HMG 1 proteins belong to the ‘‘high mobility group’’ class of mammalian chromatin proteins and are present in all vertebrate nuclei. Their sequence conservation, ubiquity and abundance suggest important functions for HMG 1. They appear to be involved in a large number of DNA transactions (reviewed by Travers et al., 1994; Bianchi, 1994). HMG 1 proteins have three structural domains: the N-terminal A-domain and the central B-domain are positively charged and bind to DNA, while the terminal C-domain is acidic and interacts with histones (general reviews and references therein; Bianchi et al., 1992a; Landsman & Bustin, 1993; Grosschedl et al., 1994). Although HMG 1 binds to double-stranded DNA and facilitates the circularization of DNA restriction fragments (Pil et al., 1993; Paull et al., 1993), it binds preferentially to bent DNA such as four-way junction DNA (Bianchi et al., 1992b), cisplatin-modified DNA (Pil & Lippard, 1992; Hughes et al., 1992) and DNA containing bulges (Pontiggia & Bianchi, unpublished results). The sequences of the two domains A and B of HMG 1 are homologous to each other (Figure 1) and to segments of about 70 amino acid residues called HMG box. The two boxes A and B of HMG 1 bind to the fourway junction DNA with the same affinity (Bianchi et al., 1992b), which is in favor of a similar mode of interaction with DNA. The data reported here strengthen this suggestion. We find that the two boxes A and B protect the same region of a cis-diamminedichloroplatinum(II) (cis-DDP)modified double-stranded oligonucleotide contain0022–2836/95/070243–05 $08.00/0
ing a single intrastrand cross-link at the d(GpG) site (abbreviated d(G*pG*)) from DNase I cleavage. In addition, we show by circular dichroism that the boxes A and B have the same folding and that they induce the same conformational changes in the platinated DNA. Recent studies (Hughes et al., 1992; Pil & Lippard, 1992) have shown that HMG 1 interacts with DNA modified with cis-DDP at d(GpG) and d(ApG) sites and does not interact with DNA modified with trans-DDP (trans-DDP is the stereoisomer of cis-DDP). The endonuclease DNase I has been extensively used to study the interactions between DNA and ligands (Tullius, 1989). Alterations induced in the DNA double helix by cis-DDP have been revealed by several approaches, including DNase I footprinting (general reviews and references therein; Kozelka & Chottard, 1990; Lepre & Lippard, 1990; Sip & Leng, 1993). The DNase I cleavage patterns of oligonucleotides bearing or not a single cis-DDP intrastrand cross-link d(G*pG*) are very different (Visse et al., 1991; Schwartz & Leng, 1994). The data in Figure 2 (lanes 0) confirm this observation. In the three studies (Visse et al., 1991; Schwartz & Leng, 1994; this work), the central sequences of the three oligonucleotides are d(CACTGGAACC), d(TCTTGGTTCT) and d(CCTCGGTTCT), respectively. The strong changes in the DNase I cleavage patterns occur mainly at the level of the 3' residue adjacent to the adduct and at the level of the C residue complementary to the platinated 5' G residue. The efficiency of the DNase I cutting appears to be 7 1995 Academic Press Limited
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Figure 1. Sequences of the boxes A and B of rat HMG 1 (identities and similarities for the aminoacids are indicated by asterisks (*) and dots, respectively) and of the oligonucleotides for the DNase I footprinting (1) and for the circular dichroism (2).
influenced by the distortions induced in the double helix by the adduct more than by the base sequence. The presence of HMG 1-box A does not affect the DNase I cleavage pattern of the unplatinated oligonucleotide while it does affect the cleavage pattern of the platinated oligonucleotide (Figure 2). Inspection of the gels shows that the box A inhibits the DNase I cutting over at least 15 and 12 nucleotide residues in the upper and lower strands, respectively. Interestingly, the protected footprints are similar for the non-sequence-specific HMG 1-box A and the sequence-specific Sox-5 HMG box (Connor et al., 1994). In the upper strand, the most noticeable effect is at the level of the T residue adjacent to the central d(GpG) site on its 3' side. This new DNase I-sensitive site after platination, is hardly cleaved in the complex with box A. In the lower strand, the most striking effect occurs at the C residue complementary to the platinated 5' G residue. At this site, the DNase I cutting, strongly enhanced by the platination, is inhibited by the presence of the box A. The next step was to compare the effects of box A and box B on the cleavage patterns of the platinated oligonucleotide. The DNase I cleavage patterns of the platinated oligonucleotide in the presence of the two boxes are identical (Figure 3); thus as judged by DNase I, the two boxes behave very similarly with respect to DNA. The crystallographic structure of the DNase I-DNA complex revealed that an exposed loop of the protein penetrates into the minor groove of the DNA and interacts asymmetrically with the backbone of both strands. DNase I contacts two phosphate groups on each side of the cleaved bond and two phosphate groups on the other strand across the minor groove (Suck & Oefner, 1986; Suck et al., 1988). The enhanced DNase I cleavage of the platinated DNA has been related to the distortions induced in DNA by the d(G*pG*) adduct (Visse et al., 1991; Schwartz & Leng, 1994). The adduct bends the double helix by 32–34° towards the major groove (and thus widens the minor groove) and unwinds it by 13° (Bellon et al., 1991). On the basis of the solution structure of the HMG box B determined by NMR (Weir et al., 1993; Read et al., 1993) and of several experiments (reviewed by Grosschedl et al., 1994; Falciola et al., 1994), a model has been proposed in which the extended segment of the HMG box binds into the
Figure 2. DNase I footprinting of the HMG 1 box A-DNA complex. The synthesis of the oligonucleotide and the platination at the d(GpG) site in the upper strand were done as described (Schwartz & Leng, 1994) with minor modifications. The protocol of Bianchi et al. (1992b) was followed to isolate the 2 boxes. They were further purified on a FPLC MonoS column. The double stranded oligonucleotides (0.3 mM) with a 5'-labeled upper strand (left) or lower strand (right) were subjected to limited DNase I digestion in 50 ml of buffer containing 20 mM Hepes (pH 8), 100 mM KCl, 7 mM MgCl2 , 1.5 mM CaCl2 , 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin, 2 mg of sonicated calf thymus DNA and 0.1 unit of DNase I (Boehringer). Similar results were obtained with 0.05 or 0.2 unit of DNase I. The digestions were carried out for 3 min at 20°C. The reactions were stopped by addition of 5 mM EGTA. The samples were loaded onto a denaturing 12% polyacrylamide gel. Maxam & Gilbert sequencing reactions were run in parallel (not shown). The concentrations of the box A were respectively 1.3 mM (lane 1), 6.3 mM (lane 2) and 13 mM (lane 3); no box A (lane 0). The arrows on each side of the sequences indicate the limits of the DNase I protected regions. The fragments containing the adducts migrate slower than the corresponding unplatinated fragments because the platinum residues were not removed before the electrophoresis.
enlarged minor groove of the platinated DNA (Faciola et al., 1994). The strong inhibition of the platinated DNA cutting by DNase I in the presence of the boxes supports the model. Assuming that in solution, a proficient incision by DNase I implies the same contacts between the phosphate groups and DNase I as those observed in the crystal (Fairall & Rhodes, 1992), one can deduce that the HMG box protects at least six nucleotide residues on each strand of the platinated DNA (rectangular box on the cylindrical projection of the DNA, Figure 3). It seems likely that the protected region extends more on the 5' side of the adduct but this cannot be guaranteed because of the absence of strong DNase I cleavage sites in the lower strand. On the other hand, an NMR study of the complex between SRY HMG box and DNA (King & Weiss, 1993) demonstrates the recognition of two A · T base-pairs of the DNA by
JMB—MS 335 Communication
Figure 3. DNase I footprinting of the platinated DNA in the presence of the HMG 1 box A or B. The experimental conditions were as described for Figure 2. The concentrations of the boxes were, respectively, 1.8 mM (lanes 1 and 2) and 5.3 mM (lanes 3 and 4). In the right part of the Figure is shown a schematic representation of the contacts between DNase I and the box-platinated DNA complex plotted on a cylindrical projection of the double helix. The base-pairs are drawn across the minor groove. An arrow ( 3 ) marks the DNase I cleavage site and the dots (,) mark the phosphate groups contacted by DNase I to cleave at the site. The minimal size of the protected region by HMG 1 box is indicated by a rectangle.
partial intercalation of a non-polar side-chain of the SRY box. Such an interaction could also occur in the HMG 1 box-DNA complex. We found (not shown) that a DNA containing a single trans-DDP interstrand cross-link is not recognized by the HMG 1 boxes, although the global distortion induced in DNA by the trans-DDP interstrand cross-link and the cis-DDP intrastrand cross-link present some resemblance. The trans-DDP interstrand cross-link bends the double helix towards the major groove by 26° and unwinds it by 12° (Brabec et al., 1993). It is tempting to speculate that the cis-DDP intrastrand cross-link makes easier the partial intercalation (the Pt residue is located in the major groove) while the trans-DDP interstrand cross-link prevents it (the Pt residue is located between the two strands). The boxes A and B have been further compared by circular dichroism (CD). The far ultra-violet spectra recorded down to 185 nm, are identical within experimental errors (Figure 4, 1). The secondary structure contents of the two boxes estimated by the unconstrained variable selection method (Manavalan & Johnson, 1987) are the same (a-helix, 56(2)1%; b-sheet, 6(2)2%; turn, 11(2)1%; other, 27(2)1%). We conclude that the two HMG 1 boxes A and B have the same folding. The values of the secondary structure contents are in good agreement with the CD values reported for the HMG 1 box B (Weir et al., 1993; Read et al., 1993), TCF-1 HMG box (van Houte et al., 1993) and Sox-5 HMG box (Connor et al., 1994). However, an intriguing point is that from
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Figure 4. Circular dichroism of the HMG 1 boxes A and B (1) and of the box-platinated DNA complexes (2). The spectra were recorded on a Jobin-Yvon spectrophotometer at room temperature. The samples were dissolved in 100 mM NaF, 10 mM phosphate buffer (pH 7.5), 0.5 mM DTT. The concentrations of the HMG boxes were 5 mM. In 2, (- · -) corresponds to the free platinated oligonucleotide, (- -) to the free HMG box A (or B) and (—) to the HMG box A (or B)-platinated oligonucleotide complex. Below 250 nm, the calculated spectrum (not shown) corresponding to the difference between the spectrum of the complex and the spectrum of the free platinated oligonucleotide resembles the spectrum of the free protein.
the NMR structure of the HMG 1 box B (Weir et al., 1993; Read et al., 1993) and of the HMG-D box (Jones et al., 1994) the a-helical content is about 75 %, which disagrees with the CD estimate. It has been argued that CD underestimates the a-helical content because of the presence of tyrosine (Bradley et al., 1990; Weir et al., 1993). This seems unlikely since the boxes A and B contain a different number of tyrosine residues and yet have the same CD spectra. This discrepancy between CD and NMR deserves to be explained. No differences were found in the CD spectra of the complexes between the platinated oligonucleotide and the box A or the box B; one of these experiments is shown in Figure 4, 2. The spectrum of the platinated oligonucleotide, slightly different from that of the unplatinated oligonucleotide in agreement with previous results (van Hemelrick et al., 1986), is modified by the binding of the box. Taking into account the concentrations of the protein and DNA, the contribution of the protein to the CD signal at wavelengths larger than 250 nm can be neglected. The modifications between the spectra of the free platinated DNA and of the complex (a decrease of the intensity and a small red-shift of the positive band ) reveal unambiguously a conformational change of the DNA induced by the binding of the HMG box. In this wavelength region, our CD results resemble those reported on HMG 1-DNA (Stros et al., 1994) but differ from those on Sox-5-DNA (Connor et al., 1994). It is premature to discuss further the conformational changes induced in three different DNAs by the binding of the HMG boxes. At wavelengths shorter than 250 nm, both the DNA and protein contribute to the CD signal and thus it is difficult to attribute unambiguously the observed changes to structural modifications of either one (or both) of the interacting molecules. Nevertheless, the fact that these variations are rather small allows exclusion of the
JMB—MS 335 246 possibility of a large conformational change of the protein, such as the formation of an a-helix described for the binding to DNA of GCN4 and the bZip fragment of C/EBP (O’Neil et al., 1991). In conclusion, the two boxes A and B of HMG 1, which have significantly different sequences, behave identically as judged by circular dichroism and DNase I footprinting. Work is in progress to characterize the interactions between the entire protein HMG 1 and DNA.
Acknowledgements We are indebted to Dr A. R. Rahmouni and A. Schwartz for helpful discussions. This work was supported in part by la Ligue contre le Cancer, la Fondation pour la Recherche Me´dicale, l’Association de la Recherche sur le Cancer and the E.E.C. (projects CHRX-CT92-0016, D1-92-002 and CHRX-CT94-0482).
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Edited by M. Yaniv
(Received 3 October 1994; accepted 21 November 1994)