Mutagenesis analysis of the interaction between the dorsal rel homology domain and HMG boxes of DSP1 protein

DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((253)KKRK(256)) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.     

The HMG domain of the ROX1 protein mediates repression of HEM13 through overlapping DNA binding and oligomerization functions.

The ROX1 gene of Saccharomyces cerevisiae encodes a protein required for the repression of genes expressed under anaerobic conditions. ROX1 belongs to a family of DNA binding proteins which contain the high mobility group motif (HMG domain). To ascertain whether the HMG domain of ROX1 is required for specific DNA binding we synthesized a series of ROX1 protein derivatives, either in vitro or in Escherichia coli as fusions to glutathione S-transferase (GST) protein, and tested them for their ability to bind to DNA. Both ROX1 proteins that were synthesized in vitro and GST-ROX1 fusion proteins containing the intact HMG domain were able to bind to specific target DNA sequences. In contrast, ROX1 proteins which contained deletions within the HMG domain were no longer capable of binding to DNA. The oligomerization of ROX1 in vitro was demonstrated using affinity-purified GST-ROXI protein and ROX1 labelled with [35S]methionine. Using various ROX1 protein derivatives we were able to demonstrate that the domain required for ROX1-ROX1 interaction resides within the N-terminal 100 amino acids which constitute the HMG domain. Therefore, the HMG domain is required for both DNA binding activity and oligomerization of ROX1.     

Characterization of high mobility group protein binding to cisplatin-damaged DNA.

cis-Diamminedichloroplatinum (II) (cisplatin, CDDP) is a widely used chemotherapeutic agent. While many tumors are highly responsive to CDDP, certain tumors are resistant to this drug, limiting its efficacy. The anti-tumor activity of CDDP is believed to result from its coordination bonding to chromosomal DNA. Alterations in tumor cell sensitivity to CDDP may result from the presence or absence of protein(s) which specifically recognize CDDP-damaged DNA. We have developed a damaged-DNA affinity precipitation assay that allows the direct identification of cellular proteins that bind to CDDP-damaged DNA. Using this procedure, we have identified several proteins which specifically bind to CDDP-damaged DNA. Two of these proteins have been identified as high mobility group proteins (HMG) 1 and 2 in the current report, we have characterized the binding of these proteins to CDDP-DNA. The calculated Kd of binding to CDDP-damaged DNA was 3.27 x 10(-10) for HMG1 and 1.87 x 10(-10) for HMG2. Using highly specific chemical modifying reagents, we have determined that Cys residues play an important role in protein binding. We also observed that HMG2 will bind to DNA modified with carboplatin and iproplatin although to a lesser extent than to DNA damaged with CDDP. Thus, our results indicate that HMG 2 binds with high affinity to DNA modified with therapeutically active platinum compounds. In addition, our findings suggest that thiol groups play an essential role in the binding of HMG1 and HMG2 to CDDP-DNA.     

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.     

HMGB1 interacts differentially with members of the Rel family of transcription factors

HMGB1 is an architectural factor that enhances the DNA binding affinity of several proteins. We have investigated the influence of HMGB1 on DNA binding by members of the Rel family. HMGB1 enhances DNA binding by p65/p50 and p50/p50, but reduces binding by p65/p65, c-Rel/c-Rel, p65/c-Rel, and p50/c-Rel. In pull-down assays, HMGB1 interacts directly with the p50 subunit via its HMG boxes and this interaction is weakened by the presence of the acidic tail. Functionally, HMGB1 is required for the NF-kappaB-dependent expression of the adhesion molecule VCAM-1.     

The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins.

The mammalian nuclear protein HMG1 contains two segments that show a high sequence similarity to each other. Each of the segments, produced separately from the rest of the protein in Escherichia coli, binds to DNA with high specificity: four-way junction DNA of various sequences is bound efficiently, but linear duplex DNA is not. Both isolated segments exists as dimers in solution, as shown by gel filtration and chemical crosslinking experiments. HMG1-like proteins are present in yeast and in protozoa: they consist of a single repetition of a motif extremely similar to the DNA binding segments of HMG1, suggesting that they too might form dimers with structural specificity in DNA binding. Sequences with recognizable similarity to either of the two DNA binding segments of HMG1, called HMG boxes, also occur in a few eukaryotic regulatory proteins. However, these proteins are reported to bind to specific sequences, suggesting that the HMG box of proteins distantly related to HMG1 might differ significantly from the HMG box of HMG1-like proteins.