FACT-mediated exchange of histone variant H2AX regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16.

The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.

HMGA Interactome: new insights from phage display technology.

High mobility group A proteins (HMGA1 and HMGA2) are architectural factors involved in chromatin remodelling and regulation of gene expression. HMGA are highly expressed during embryogenesis and in cancer cells and are involved in development and cell differentiation as well as cancer formation and progression. These factors, by binding to DNA and interacting with other nuclear proteins, can organize macromolecular complexes involved in transcription, chromatin dynamics, RNA processing, and DNA repair. The identification of protein partners for HMGA has greatly contributed to our understanding of their multiple functions. He we report the identification of HMGA molecular partners using a gene fragment library in a phage display screening. Using an ORF-enriched cDNA library, we have isolated several HMGA1 interacting clones and for two of them, TBP associated factor 3 (TAF3) and chromatin assembly factor 1 p150/CAF-1, have demonstrated an in vivo association with HMGA1. The identification of these new partners suggests that HMGA can also influence general aspects of transcription and once more underlines their involvement in chromatin remodelling and dynamics.

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing.

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the 'ruler' that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

Tetrahymena contain two distinct and unusual high mobility group (HMG)-like proteins.

Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K. Iwai, 1979, J. Biochem. [Tokyo], 69:1097-1111; Levy-Wilson, B., M. S. Denker, and E. Ito, 1983, Biochemistry, 22:1715-1721). In this report, two of these proteins, LG-1 and LG-2, have been further characterized. Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses. As well, LG-1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins. Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17. Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins. Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C. D. Allis, R. Richman, R. G. Cook, and M. A. Gorovsky, 1986, Proc. Natl. Acad. Sci. USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein. Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schröter, H., G. Maier, H. Ponsting, and A. Nordheim, 1985, Embo (Eur. Mol. Biol. Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes. Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype. However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h. Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1. Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific recognition of cruciform DNA by nuclear protein HMG1.

Cruciform DNA, a non-double helix form of DNA, can be generated as an intermediate in genetic recombination as well as from palindromic sequences under the effect of supercoiling. Eukaryotic cells are equipped with a DNA-binding protein that selectively recognizes cruciform DNA. Biochemical and immunological data showed that this protein is HMG1, an evolutionarily conserved, essential, and abundant component of the nucleus. The interaction with a ubiquitous protein points to a critical role for cruciform DNA conformations.

Expression of a high mobility group protein isolated from Cucumis sativus affects the germination of Arabidopsis thaliana under abiotic stress conditions.

Although high mobility group B (HMGB) proteins have been identified from a variety of plant species, their importance and functional roles in plant responses to changing environmental conditions are largely unknown. Here, we investigated the functional roles of a CsHMGB isolated from cucumber (Cucumis sativus L.) in plant responses to environmental stimuli. Under normal growth conditions or when subjected to cold stress, no differences in plant growth were found between the wild-type and transgenic Arabidopsis thaliana overexpressing CsHMGB. By contrast, the transgenic Arabidopsis plants displayed retarded germination compared with the wild-type plants when grown under high salt or dehydration stress conditions. Germination of the transgenic plants was delayed by the addition of abscisic acid (ABA), implying that CsHMGB affects germination through an ABA-dependent way. The expression of CsHMGB had affected only the germination stage, and CsHMGB did not affect the seedling growth of the transgenic plants under the stress conditions. The transcript levels of several germination-responsive genes were modulated by the expression of CsHMGB in Arabidopsis. Taken together, these results suggest that ectopic expression of a CsHMGB in Arabidopsis modulates the expression of several germination-responsive genes, and thereby affects the germination of Arabidopsis plants under different stress conditions.

Purification and characterization of a high-mobility-group-like DNA-binding protein that stimulates rRNA synthesis in vitro.

A 16,000-dalton, high-mobility-group-like (HMG-like) DNA-binding protein, referred to as p16, has been purified to homogeneity from Novikoff hepatoma ascites cells. p16 binds specifically to a portion of the 5' flanking region of the rat rRNA gene (-620 to -417), which is part of the upstream activator sequence identified previously (B. G. Cassidy, H.-F. Yang-Yen, and L. I. Rothblum, Mol. Cell. Biol. 6:2766-2773, 1986). p16 also binds to a segment of the external transcribed spacer (+352 to +545). In vitro reconstituted transcription experiments demonstrated that the addition of p16 stimulated rRNA synthesis up to ca. fourfold. The stimulation was dose dependent and saturable. The effect of p16 on ribosomal gene transcription was also dependent on the presence of either the upstream or the downstream DNA-binding site, or both. The amino acid composition of p16 is very similar to that of HMG-I, suggesting that p16 may be a member of the HMG-I family of proteins. In this case, our results suggest that HMG proteins may play an important role in the regulation of the rRNA gene expression.

The testis-specific high-mobility-group protein, a phosphorylation-dependent DNA-packaging factor of elongating and condensing spermatids.

Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines. The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA. A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process. We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids. Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro. The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner. We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG. Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I. This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements. When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs). Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences. Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays. This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding. Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR. The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA. The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold. Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE. HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences. The A- or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding. DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure. It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form. The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA.

Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process.

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.

A high-mobility-group protein and its cDNAs from Drosophila melanogaster.

We have identified, purified, and characterized a high-mobility-group (HMG) protein and its cDNAs from Drosophila melanogaster. This protein, HMG D, shares most of the characteristics of vertebrate HMG proteins; it is extractable from nuclei with 0.35 M NaCl, is soluble in 5% perchloric acid, is relatively small (molecular weight of 12,000), has both a high basic (24%) and high acidic (24%) amino acid content, and is a DNA-binding protein. HMG D exhibits characteristics of both the vertebrate HMG 1 and 2 class and the HMG 14 and 17 class of proteins. Its amino acid sequence is similar (36% amino acid identity) to that of HMG1, while its size and selective extraction with ethidium bromide are similar to properties of the HMG 14 and 17 class of proteins. HMG D is encoded by a single-copy gene that maps to 57F8-11 on the right arm of chromosome 2. Two transcripts are observed during embryogenesis; the protein is relatively stable throughout development. By the biochemical criteria of size, solubility, and amino acid content, HMG D appears to be the major HMG protein of D. melanogaster.

Regulation of cell-type-specific interleukin-2 receptor alpha-chain gene expression: potential role of physical interactions between Elf-1, HMG-I(Y), and NF-kappa B family proteins.

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is rapidly and potently induced in T cells in response to mitogenic stimuli. Previously, an inducible enhancer between nucleotides -299 and -228 that contains NF-kappa B and CArG motifs was identified. We now report the characterization of a second essential positive regulatory element located between nucleotides -137 and -64 that binds Elf-1 and HMG-I(Y). This element had maximal activity in lymphoid cells, paralleling the cell type specificity of Elf-1 expression. Transcription from the IL-2R alpha promoter was inhibited when either the Elf-1 or the HMG-I(Y) binding site was mutated. Coexpression of both proteins activated transcription of the -137 to -64 element in COS-7 cells. Elf-1 physically associated with HMG-I and with NF-kappa B p50 and c-Rel in vitro, suggesting that protein-protein interactions might functionally coordinate the actions of the upstream and downstream positive regulatory elements. This is the first report of a physical interaction between an Ets family member and NF-kappa B family proteins. These findings provide significant new insights into the protein-protein and protein-DNA interactions that regulate cell-type-specific and inducible IL-2R alpha gene expression and also have implications for other genes regulated by Elf-1 and NF-kappa B family proteins.

HMG-I(Y) recognizes base-unpairing regions of matrix attachment sequences and its increased expression is directly linked to metastatic breast cancer phenotype.

Base-unpairing regions (BURs) contain a specialized DNA context with an exceptionally high unwinding propensity, and are typically identified within various matrix attachment regions. A BUR affinity column was used to purify a doublet of Mr 20,000 proteins from human breast carcinoma cells. These proteins were identified as the high-mobility group (HMG) protein, HMG-I, and its splicing variant, HMG-Y. We show that HMG-I(Y) specifically binds BURs. Mutating BURs so as to abrogate their unwinding property greatly reduced their binding affinity to HMG-I(Y). Numerous studies have indicated that elevated HMG-I(Y) expression is correlated with more advanced cancers and with increased metastatic potential. We studied whether the expression of HMG-I(Y) responds to signaling through the heregulin (HRG)-erbB pathway and the extracellular matrix. HMG-I(Y) expression was increased in MCF-7 cells after stable transfection with an HRG expression construct that led cells to acquire estrogen independence and metastasizing ability. A high level of HMG-I(Y) expression was detected in metastatic MDA-MB-231 cells, but the expression was virtually diminished, and the metastasizing ability was lost after cells were stably transfected with an antisense HRG cDNA construct. HMG-I(Y) was also decreased in MDA-MB-231 cells when treated with a chemical inhibitor for matrix metalloproteinase-9 that led to a reduction of invasive capability in vitro. The level of HMG-I(Y) expression, therefore, is dynamically regulated in human breast cancer cells in response to varying types of signaling that affect metastatic ability, including the HRG-erbB pathway and those from the extracellular matrix.

Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2.

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.

Reexamination of the high mobility group-1 protein for self-association and characterization of hydrodynamic properties.

Previous studies of the 25 kDa high mobility group-1 (HMG-1) protein have generated conflicting results regarding whether HMG-1 exists as a monomer or is capable of oligomerizing to (functional) tetramers. To resolve this question, sedimentation velocity analysis yielded a s20,w value of 2.59S, which is consistent with a monomeric protein. Equilibrium sedimentation data were obtained for three HMG-1 concentrations at two rotor speeds. The six sets of data were fit to both an ideal single component and monomer-dimer equilibrium model, with essentially identical fits produced for both models, with the latter indicating a low extent (7%) of dimerization. Reaction of HMG-1 with glutaraldehyde produced a small population of oligomers consistent with a low level of dimers. This supported the monomer-dimer equilibrium model. Surprisingly, gel permeation chromatography yielded an apparent molecular mass of approx. 55 kDa for both HMG-1 and HMG-2. This finding is considered anomalous and presumably due to the high negative charge density in the C terminus of HMG-1. The sedimentation data also permit one to model HMG-1 as a hydrated prolate ellipsoid with a major axis/minor axis ratio of 2. 79. The collective evidence from the sedimentation and chemical cross-linking studies strongly supports a moderately asymmetric monomer in solution and unequivocally eliminates the possibility of a highly extended shape for HMG-1 or the existence of any extensive oligomerization.

Physical properties of chicken erythrocyte HMG-1, HMG-2 and HMG-E.

HMG-1, HMG-2 and HMG-E were purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions and subjected to several types of physical measurement. The principal conclusions drawn from the measurements were: none of the proteins has a strong tendency to self-associate, although HMG-1 does weakly self-associate; the frictional properties of HMG-1 and HMG-E (and probably HMG-2) indicate that the proteins deviate significantly from compact, moderately hydrated spheres; and each of the proteins contains approximately 40% helix and little if any beta-pleated sheet.

Isolation, characterisation and growth-related changes of an HMG-like protein from microplasmodia of Physarum polycephalum.

An alanine, lysine and glutamic acid-rich nuclear protein (P2) of Mr approximately 19,500 co-extracts with the histones from nuclei of Physarum polycephalum when using the CaCl2 method for histone extraction [1] and was found to have the composition previously ascribed to a putative histone H1(0) isolated from microplasmodia using 5% PCA (Yasuda, H., Mueller, R.D., Logan, K.A. and Bradbury, E.M. (1986) J. Biol. Chem. 261, 2349-2354). P2 has very similar electrophoretic properties to chicken erythrocyte histone H5, calf thymus histone H1(0) and the Physarum HMG-like protein AS-2, but does not appear to be immunologically or structurally similar to H5 or H1(0). An increase in the abundance of P2 was observed during exponential growth in microplasmodia, reaching an approximately 1:1 ratio with histone H1 by 48 h of culture. Standard amino acid analysis and NMR show that P2 is more HMG-like than H1-like and CD measurements demonstrated that P2 contains only 5% secondary structure in its maximally structured state and is, therefore, essentially unstructured under in vivo conditions. Also possible clustering of acidic residues is detected using CD and may be of functional significance. Analysis of post-translational modification of P2 shows that it is phosphorylated at up to three sites as isolated from immature spherules. The relationship of P2 to the HMG family of proteins and AS-2 is discussed.

Binding of high-mobility-group proteins HMG 14 and HMG 17 to DNA and histone H1 as influenced by phosphorylation.

We have used affinity chromatography to study the effects of phosphorylation of calf thymus high-mobility-group proteins HMG 14 and HMG 17 on their binding properties towards calf thymus single- and double-stranded DNA and histone H1. Without in vitro phosphorylation, HMG 14 and HMG 17 eluted from double-stranded DNA-columns at 200 mM NaCl. HMG 14 was released from single-stranded DNA-column at 300 mM NaCl and from H1-column at 130 mM NaCl, whereas the corresponding values for HMG 17 were 230 mM and 20 mM, respectively. Phosphorylation of HMG 14 and HMG 17 by cAMP-dependent protein kinase (A-kinase) decreased markedly their affinity (270 mM and 200 mM NaCl, respectively) for single-stranded DNA, whereas HMG 14 phosphorylated by nuclear protein kinase II (NII-kinase) eluted only slightly (290 mM NaCl) ahead of the unphosphorylated protein. HMG 14 phosphorylated by both A-kinase and NII-kinase eluted from double-stranded DNA-columns almost identically (190 mM NaCl) with the unphosphorylated protein. Interestingly, phosphorylation of HMG 14 by NII-kinase increased considerably its affinity for histone H1 and the phosphorylated protein eluted at 200 mM NaCl. Phosphorylation of HMG 14 by A-kinase did not alter its interaction towards histone H1. These results indicate that modification of HMG 14 by phosphorylation at specific sites may have profound effects on its binding properties towards DNA and histone H1, and that HMG 17 has much weaker affinity for single-stranded DNA and histone H1 than HMG 14.

Interaction of HMG14 with chromatin.

Neutron scattering has been used to study the interaction of HMG14 with chromatin. Chromatin depleted of H1/H5 was reconstituted separately with histones H1 and H5, and complexed with HMG14. We have also studied the conformation of complexes formed by the binding of HMG14 to nucleosome dimers without linker DNA. Our data on the binding of HMG14 to linkerless nucleosome dimers argue against a significant change in the exit and entry angles of nucleosomal core DNA. Data on the condensation of chromatin into a higher-order structure suggest that there is no dramatic difference between the roles of H1 and H5 in their influence on HMG14 complex formation. However, there is a decrease of about 25% in the mass per unit length of chromatin fibers on HMG14 binding, which is not accompanied by a change in the fiber repeat distance. This is evidence that there are fewer nucleosomes per repeat in HMG14 containing chromatin fibers than in normal chromatin. Alteration of chromatin structure in this manner may be part of the role of HMG14 in actively transcribed chromatin.

Interaction between cisplatin-modified DNA and the HMG boxes of HMG 1: DNase I footprinting and circular dichroism.

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 alpha-helical content (56%) and they induce the same conformational changes in the platinated DNA.