High Mobility Group Domain Proteins Research Articles

Microsporidia Evolved from Ancestral Sexual Fungi

Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested microsporidia share a common ancestor with fungi [2-7]. However, the exact nature of this phylogenetic relationship is unclear because of unusual features of microsporidial genomes, which are compact with fewer and highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, or whether microsporidia are a sister group to all fungi. Here, we present evidence addressing this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster governing sexual reproduction in which a high mobility group (HMG) transcription-factor gene is flanked by triose-phosphate transporter (TPT) and RNA helicase genes [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order. Genome-wide synteny analysis reveals multiple other loci conserved between microsporidia and zygomycetes to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor and suggest microsporidia may have an extant sexual cycle.

Mitochondrial Transcription Factor A Induction by Redox Activation of Nuclear Respiratory Factor 1

The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondria-rich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network.

N-Ethyl- and [N,N‘-Diethyl-1,2-bis(2,6-difluoro-3-hydroxyphenyl)- ethylenediamine]dichloroplatinum(II): Structure and Cytotoxic/Estrogenic Activity in Breast Cancer Cells

N-Ethyl and N,N'-diethyl derivatives (erythro- and threo-2-PtCl2; meso- and D,L-3-PtCl2) of [meso- and [D,L-1,2-bis(2,6-difluoro-3-hydroxyphenyl)ethylenediamine]dichloroplatinum(II) (meso- and D,L-1-PtCl2) were synthesized and tested for cytotoxicity on the estrogen receptor-positive (ER+) human MCF-7 breast cancer cell line. In this test, only D,L-1-PtCl2 and threo-2-PtCl2 showed strong cytotoxic properties. This revealed the existence of at least one NH2 fragment as a prerequisite for antitumor activity. Furthermore, studies on the three-dimensional structure of the new compounds demonstrated that the aryl and alkyl residues at the five-membered chelate ring have to be arranged in equatorial positions for the triggering of cytotoxic effects, very likely due to the reaction with d(GpG) sequences in DNA resulting in GG-N7,N7 chelates. A contribution of the ER-mediated processes--(a) hindrance of the cellular processing of Pt-modified DNA by overexpression of high mobility group domain proteins and (b) interruption of the vicious circle of mutual growth stimulation of breast cancer cells and granulocytes/macrophages by reduction of the formation of key cytokines--to the anti-breast cancer activity of threo-2-PtCl2 is unlikely, since we did not observe transcription activation in the test on ER+ MCF-7 breast cancer cells stably transfected with luciferase reporter plasmid ERE(wtc)luc.

Mint Represses Transactivation of the Type II Collagen Gene Enhancer through Interaction with αA-crystallin-binding Protein 1

Collagen type II is an extracellular matrix protein important for cartilage and bone formation, and its expression is controlled by multiple cis- and trans-acting elements, including the zinc finger transcription factor alpha A-crystallin-binding protein 1 (CRYBP1). Here we show that MSX2-interacting nuclear target protein (MINT), a conserved transcriptional repressor, associates with CRYBP1 and negatively regulates the transactivation of the collagen type II gene (Col2a1) enhancer. We identified CRYBP1 as a binding partner of MINT by screening a mouse embryonic cDNA library using the yeast two-hybrid system. We demonstrated that the C terminus of MINT interacts with the C terminus of CRYBP1 using the mammalian cell two-hybrid assay, glutathione S-transferase pull-down, and co-immunoprecipitation analyses. Furthermore, MINT and CRYBP1 form a complex on the Col2a1 enhancer, as shown by chromatin immunoprecipitation and gel shift assays. In the presence of CRYBP1, overexpression of MINT or its C-terminal fragment in cells repressed a reporter construct driven by the Col2a1 enhancer elements. This transcription repression is dependent on histone deacetylase, the main co-repressor recruited by MINT. The present study shows that MINT is involved in CRYBP1-mediated Col2a1 gene repression and may play a role in regulation of cartilage development.

The High-Mobility-Group Domain Transcription Factor Rop1 Is a Direct Regulator of prf1 in Ustilago maydis

In the smut fungus Ustilago maydis, the pheromone signal is transmitted via a mitogen-activated protein kinase module to the high-mobility-group (HMG) domain transcription factor Prf1, leading to its activation. This triggers sexual and pathogenic development since Prf1 binds to the PRE boxes located in the promoters of the a and b mating type genes. Here, we present the characterization of rop1 and hmg3, encoding two additional sequence-specific HMG domain proteins. While hmg3 mutants are slightly impaired in mating and do form conjugation hyphae, rop1 deletion strains display a severe mating and filamentation defect and do not respond to pheromone stimulation. In particular, rop1 is essential for pheromone-induced gene expression in axenic culture. Constitutive expression of prf1 fully complements the mating defect of rop1 mutants, indicating that rop1 is required for prf1 gene expression. Indeed, we could show that Rop1 binds directly to specific elements in the prf1 promoter. Surprisingly, on the plant surface, rop1 deletion strains do form conjugation hyphae and express sufficient amounts of prf1 to cause full pathogenicity. This indicates the involvement of additional components in the regulation of prf1 gene expression during pathogenic growth.

Cisplatin Resistance and Transcription Factors

Cisplatin is one of the most potent and widely used anti-cancer agents in the treatment of various solid tumors. However, the development of resistance to cisplatin is a major obstacle in clinical treatment. Several mechanisms are thought to be involved in cisplatin resistance, including decreased intracellular drug accumulation, increased levels of cellular thiols, increased nucleotide excision-repair activity and decreased mismatch-repair activity. In general, the molecules responsible for each mechanism are upregulated in cisplatin-resistant cells; this indicates that the transcription factors activated in response to cisplatin might play crucial roles in drug resistance. It is known that the tumor-suppressor proteins p53 and p73, and the oncoprotein c-Myc, which function as transcription factors, influence cellular sensitivity to cisplatin. So far, we have identified several transcription factors involved in cisplatin resistance, including Y-box binding protein-1 (YB-1), CCAAT-binding transcription factor 2 (CTF2), activating transcription factor 4 (ATF4), zinc-finger factor 143 (ZNF143) and mitochondrial transcription factor A (mtTFA). Two of these-YB-1 and ZNF143-lack the high-mobility group (HMG) domain and can bind preferentially to cisplatin-modified DNA in addition to HMG domain proteins or DNA repair proteins, indicating that these transcription factors may also participate in DNA repair. In this review, we summarize the mechanisms of cisplatin resistance and focus on transcription factors involved in the genomic response to cisplatin.

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder.

Protein Kinase CK2 Phosphorylates the High Mobility Group Domain Protein SSRP1, Inducing the Recognition of UV-damaged DNA

The structure-specific recognition protein SSRP1 plays a role in transcription and replication in the chromatin context. Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures. Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize SSRP1 by protein kinase CK2 alpha. The kinase phosphorylated several amino acid residues in the C-terminal part of the SSRP1 protein. Two phosphorylation sites were mapped in the very C-terminal region next to the HMG box domain, and about seven sites are localized within the acidic domain. Circular dichroism showed that the phosphorylation of the two C-terminal sites by CK2 alpha resulted in a structural change in the region of HMG box domain, because the negative peak of the CD spectrum at 222 nm was decreased by approximately 10%. In parallel, the phosphorylation induced the recognition of UV-damaged DNA, whereas the non-phosphorylated protein does not discriminate between UV-damaged DNA and control DNA. The affinity of CK2 alpha-phosphorylated SSRP1 for the DNA correlates with the degree of UV-induced DNA damage. Moreover, maize SSRP1 can restore the increased UV-sensitivity of a yeast strain lacking the NHP6A/B HMG domain proteins to levels of the control strain. Collectively, these findings indicate a role for SSRP1 in the UV response of eukaryotic cells.

Sex, Sox, and splicing

Mammalian sex-determining factors with homology to DNA binding proteins are required for splicing, according to a study by Paolo Sassone-Corsi and coworkers at the Universite Louis Pasteur (Strasbourg, France). Examination of high mobility group (HMG) domain containing SRY and Sox proteins demonstrated a surprising but clear association with splicing complexes. Figure SRY (red) colocalizes with a splicing factor (green) in nuclear speckles. SRY, the testis-determining factor found on the Y-chromosome in mice and humans, is one of several Sox family members involved in sexual development. It is known to bind DNA, in support of the proposal that HMG domain proteins act as architectural facilitators for building transcription complexes. Sassone-Corsi's group thus expected to find Sox proteins colocalized in the nucleus with transcription factors. Instead, SRY and SOX6 proteins associated with splicing factors in nuclear speckle domains. Depletion of SOX6 in HeLa cell extracts blocked splicing of multiple substrates, and expression of the HMG domain only of either SOX6, SOX9, or SRY restored splicing in the extracts, indicating functional overlap of the proteins. The group's results provide the first association between splicing and sex determination in mammals. The Drosophila genes transformer and sex-lethal encode mRNA splicing factors required for sex determination in flies, indicating that regulated splicing of sex-determining factors may be evolutionarily conserved. According to Sassone-Corsi, to complete the connection “the next step is to identify natural physiological substrates of the Sox and SRY proteins.” He thinks the Sox proteins may control regulated splicing by determining which spliceosomal complexes are formed in different cell types, analogous to the way transcription factors can regulate the basal transcription machinery. ▪ Reference: Ohe, K., et al. 2002. Proc. Natl. Acad. Sci. USA. 99:1146–1151. [PubMed] SRY (red) colocalizes with a splicing factor (green) in nuclear speckles. Sassone-Corsi/NAS

Conformation, Recognition by High Mobility Group Domain Proteins, and Nucleotide Excision Repair of DNA Intrastrand Cross-links of Novel Antitumor Trinuclear Platinum Complex BBR3464

The new antitumor trinuclear platinum compound [(trans-PtCl(NH(3))(2))(2)mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)](4+) (designated as BBR3464) is currently in phase II clinical trials. DNA is generally considered the major pharmacological target of platinum drugs. As such it is of considerable interest to understand the patterns of DNA damage. The bifunctional DNA binding of BBR3464 is characterized by the rapid formation of long range intra- and interstrand cross-links. We examined how the structures of the various types of the intrastrand cross-links of BBR3464 affect conformational properties of DNA, and how these adducts are recognized by high mobility group 1 protein and removed from DNA during in vitro nucleotide excision repair reactions. The results have revealed that intrastrand cross-links of BBR3464 create a local conformational distortion, but none of these cross-links results in a stable curvature. In addition, we have observed no recognition of these cross-links by high mobility group 1 proteins, but we have observed effective removal of these adducts from DNA by nucleotide excision repair. These results suggest that the processing of the intrastrand cross-links of BBR3464 in tumor cells sensitive to this drug may not be relevant to its antitumor effects. Hence, polynuclear platinum compounds apparently represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues.

Sequence-specific High Mobility Group Box Factors Recognize 10–12-Base Pair Minor Groove Motifs

Sequence-specific High Mobility Group Box Factors Recognize 10–12-Base Pair Minor Groove Motifs

DNA-interactions and nuclear localisation of the chromosomal HMG domain protein SSRP1 from maize.

The structure-specific recognition protein 1 (SSRP1) is a member of the protein family containing a high mobility group (HMG) domain DNA-binding motif. We have functionally characterised the 71.4 kDa Zm-SSRP1 protein from maize. The chromatin-associated Zm-SSRP1 is detected by immunoblot analysis in maize leaves, kernels and suspension culture cells, but not in roots. Mediated by its HMG domain, recombinant Zm-SSRP1 interacts structure-specifically with supercoiled DNA and DNA minicircles when compared with linear DNA. In linear duplex DNA, the protein does not recognise a specific sequence, but it binds preferentially to sequences containing the deformable dinucleotide TG, as demonstrated by a random oligonucleotide selection experiment. Zm-SSRP1 modulates DNA structure by bending the target sequence, since it promotes the circularisation of short DNA fragments in the presence of DNA ligase. Moreover, Zm-SSRP1 facilitates the formation of nucleoprotein structures, as measured using the bacterial site-specific beta-mediated recombination reaction. Analysis of the subcellular localisation of various SSRP1-GFP fusions revealed that, in contrast to HMG domain transcription factors, the nuclear localisation sequence of Zm-SSRP1 is situated within a 20-amino acid residue region adjacent to the HMG domain rather than within the DNA-binding domain. The results are discussed in the context of the likely function of SSRP1 proteins in transcription and replication.

Sequence Specificity, Conformation, and Recognition by HMG1 Protein of Major DNA Interstrand Cross-links of Antitumor Dinuclear Platinum Complexes

Interactions of high mobility group (HMG) domain proteins with DNA modified by cisplatin plays a role in mechanisms underlying its antitumor activity. A structural motif recognized by HMG domain proteins on cisplatin-modified DNA is a stable, directional bend of the helix axis. In the present work, bending induced in DNA by major adducts of a novel class of antitumor compounds, represented by the formula [¿trans-PtCl(NH(3))(2)¿H(2)N(CH(2))(2-6)NH(2)]Cl(2), was investigated. The oligodeoxyribonucleotide duplexes containing various site-specific interstrand cross-links of these bifunctional dinuclear platinum drugs were purified and characterized by Maxam-Gilbert footprinting, chemical probing, and phasing assay. It was demonstrated that the cross-links of the dinuclear compounds bent the helix much less than those of cisplatin. Gel retardation assay revealed very weak recognition of DNA adducts of dinuclear complexes by HMG1 protein. Hence, the mediation of antitumor properties of dinuclear platinum complexes by HMG domain proteins is unlikely so that polynuclear platinum compounds may represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues. A further understanding of how polynuclear platinum compounds modify DNA and how these modifications are processed in cells should provide a rational basis for the design of new platinum drugs rather than searching for cisplatin analogues.

The structure of a chromosomal high mobility group protein-DNA complex reveals sequence-neutral mechanisms important for non-sequence-specific DNA recognition.

The high mobility group (HMG) chromosomal proteins, which are common to all eukaryotes, bind DNA in a non-sequence-specific fashion to promote chromatin function and gene regulation. They interact directly with nucleosomes and are believed to be modulators of chromatin structure. They are also important in V(D)J recombination and in activating a number of regulators of gene expression, including p53, Hox transcription factors and steroid hormone receptors, by increasing their affinity for DNA. The X-ray crystal structure, at 2.2 A resolution, of the HMG domain of the Drosophila melanogaster protein, HMG-D, bound to DNA provides the first detailed view of a chromosomal HMG domain interacting with linear DNA and reveals the molecular basis of non-sequence-specific DNA recognition. Ser10 forms water-mediated hydrogen bonds to DNA bases, and Val32 with Thr33 partially intercalates the DNA. These two 'sequence-neutral' mechanisms of DNA binding substitute for base-specific hydrogen bonds made by equivalent residues of the sequence-specific HMG domain protein, lymphoid enhancer factor-1. The use of multiple intercalations and water-mediated DNA contacts may prove to be generally important mechanisms by which chromosomal proteins bind to DNA in the minor groove.

The recognition of distorted DNA structures by HMG-D: a footprinting and molecular modelling study

The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. In this study we have investigated the interaction of HMG-D, a Drosophilacounterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. We show by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, we modelled the HMG-D - bulged DNA complex. This model predicts that two residues, Val32 and Thr33, in the loop between α-helices I and II are inserted deep into the “hole” in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirmed that both are required for the efficient binding and bending of DNA by HMG-D. We discuss both the role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins and that of the basic tail in stabilising the induced DNA bend.

The High Mobility Group Domain Protein Cmb1 of Schizosaccharomyces pombe Binds to Cytosines in Base Mismatches and Opposite Chemically Altered Guanines

The mismatch-binding activity Cmb1 of Schizosaccharomyces pombe was enriched from wild type cells, and N-terminal sequencing enabled cloning of the respective gene. The deduced amino acid sequence of cmb1(+) contains a high mobility group domain, a motif that is common to a heterogeneous family of DNA-binding proteins. In crude protein extracts of a cmb1 gene-disruption strain, specific binding to C/T, C/A, and C/Delta was abolished. Weak binding to C/C revealed the presence of a second mismatch-binding activity, Cmb2. Cmb1, enriched from S. pombe and purified from Escherichia coli, bound specifically to C/C, C/T, C/A, T/T, and C/Delta but showed little or no affinity to other mismatches and small loops. Cmb1 recognizes 1,2 GpG intrastrand cross-links, produced by the chemotherapeutic drug cisplatin, when two cytosines are opposite the cross-linked guanines but not when other bases are present. Consistently, O6-methylguanine:C but not O6-methylguanine/T lesions were bound. Thus, cytosines in mismatches and opposite chemically modified guanines are the preferred target of Cmb1 recognition. cmb1 mutant cells are more sensitive to cisplatin than wild type cells, indicating a role of Cmb1 in repair of cisplatin-induced DNA damage.

Three High Mobility Group-like Sequences within a 48-Base Pair Enhancer of the Col2a1 Gene Are Required for Cartilage-specific Expression in Vivo

To understand the molecular mechanisms by which mesenchymal cells differentiate into chondrocytes, we have used the gene for an early and abundant marker of chondrocytes, the mouse pro-alpha1(II) collagen gene (Col2a1), to delineate a minimal sequence needed for chondrocyte-specific expression and to identify the DNA-binding proteins that mediate its activity. We show here that a 48-base pair (bp) Col2a1 intron 1 sequence specifically targets the activity of a heterologous promoter to chondrocytes in transgenic mice. Mutagenesis studies of this 48-bp element identified three separate sites (sites 1-3) that were essential for its chondrocyte-specific enhancer activity in both transgenic mice and transient transfections. Mutations in sites 1 and 2 also severely inhibited the chondrocyte-specific enhancer activity of a 468-bp Col2a1 intron 1 sequence in vivo. SOX9, an SRY-related high mobility group (HMG) domain transcription factor, was previously shown to bind site 3, to bend the 48-bp DNA at this site, and to strongly activate this 48-bp enhancer as well as larger Col2a1 enhancer elements. All three sites correspond to imperfect binding sites for HMG domain proteins and appear to be involved in the formation of a large chondrocyte-specific complex between the 48-bp element, Sox9, and other protein(s). Indeed, mutations in each of the three HMG-like sites of the 48-bp element, which abolished chondrocyte-specific expression of reporter genes in transgenic mice and in transiently transfected cells, inhibited formation of this complex. Overall our results suggest a model whereby both Sox9 and these other proteins bind to several HMG-like sites in the Col2a1 gene to cooperatively control its expression in cartilage.

The Drosophila fish-hook gene encodes a HMG domain protein essential for segmentation and CNS development.

We describe the isolation and analysis of the Drosophila fish-hook (fish) gene, which encodes a novel member of the SOX subgroup of High Mobility Group (HMG) domain proteins that exhibit similarity to the mammalian testis determining factor, SRY. The fish gene is initially expressed in a pair-rule-like pattern which is rapidly replaced by strong neuroectoderm expression. fish null mutants exhibit severe segmentation defects, including loss and/or fusion of abdominal denticle belts and stripe-specific defects in pair-rule and segment polarity gene expression.fish mutant embryos also exhibit loss of specific neurons, fusion of adjacent ventral nerve cord ganglia and aberrant axon scaffold organization. These results indicate an essential role for fish in anterior/posterior pattern formation and nervous system development, and suggest a potential function in modulating the activities of gap and pair-rule proteins.

Photoreactivity of platinum(II) in cisplatin-modified DNA affords specific cross-links to HMG domain proteins.

Cisplatin-modified DNA forms specific complexes with proteins that contain the DNA binding motif known as the high-mobility group (HMG) domain. As a tool for investigating the role of these proteins in mediating the cytotoxic effects of cisplatin, a set of cisplatin analogs was prepared in which one of the ammine ligands was replaced with a photoreactive tethered aryl azide ligand. The ability of DNA modified by these platinum complexes to photo-cross-link to HMG1 was investigated. During this study, it was discovered that DNA modified with cisplatin itself can undergo photoinduced cross-linking to HMG1 when irradiated with 300 nm light. The covalent complexes resulting from this latter cross-linking reaction are completely reversed by the addition of sodium cyanide and can be degraded by proteinase K. These results confirm the presence of a protein-DNA cross-link and demonstrate that the platinum atom itself forms the point of attachment. By contrast, DNA modified with transdiamminedichloroplatinum(II), [Pt(dien)Cl]Cl, or [Pt(NH3)3Cl]Cl does not cross-link to HMG1 upon irradiation. The photochemistry was exploited to cross-link a 15-base pair oligonucleotide containing a single, site-specific cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand adduct to domain B of HMG1. Following proteolytic digestion of the resulting covalent complex, the site of attachment to the protein was determined by Edman degradation of the resulting peptide-DNA complex to be a single residue on HMG domain B, Lys-6. The data further suggest that this amino acid binds to platinum at a site made available by photolabilization of a purine ligand. These results afford the first structural information about the interaction of HMG domain proteins with cisplatin-modified DNA.

A Single HMG Domain in High-Mobility Group 1 Protein Binds to DNAs as Small as 20 Base Pairs Containing the Major Cisplatin Adduct

Proteins containing a relatively new DNA-binding motif known as the high-mobility group (HMG) domain bind specifically to DNA modified by the anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Lippard, 1995). Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate that the HMG domains A and B of HMG1 are responsible for its specific interactions with cisplatin-modified DNA. An 81 amino acid recombinant protein representing a single HMG motif, HMG1 domain B, binds with an affinity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-[Pt(NH3)2-[d(GpG)-N7(1), -N7(2)]] intrastrand cross-link, at a specific site. The isolated HMG domain B binds with comparable affinity to cisplatin-modified DNAs having as few as 20 bp. The related human mitochondrial HMG domain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modulating binding of this class of proteins to cisplatin-modified DNA. This work provides direct biochemical evidence in support of conclusions drawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisplatin-modified DNA.