Regulation of gene expression depends on the integrated function of a diverse array of regulatory proteins including sequence-specific transcription factors, co-activators, and chromosomal proteins. DNA-binding chromosomal proteins can modulate the affinity and specificity of transcription factors and recruit gene regulatory machinery to specific genes for tight control of gene expression. Despite their importance in gene regulation, the chromosomal proteins have been difficult to study at the molecular level because of their characteristic ability to bind to many different sequences with comparable affinities. Consequently, the current understanding of their structures and interaction with DNA lags substantially behind that of their sequence-specific counterparts. A long term goal of this research is to understand the functions of sequence-tolerant chromosomal proteins and their interactions with DNA, and other proteins at the level of molecular structure. HMG-D is a Drosophila melanogaster member of the HMG1 family of chromosomal High Mobility Group proteins, implicated in a diverse array of functions. The HMG-box is a DNA binding motif that transcription factors use to recognize DNA sequence. Although two structures of the transcription factor HMG-box proteins bound to DNA are known, no structures of chromosomal HMG-box-DNA complexes are known. Co-crystals of the complex of DNA and HMG-D diffract to 1.8Angstrom, and native data have been collected to 2.0Angstrom. Co-crystals of HMG-D with iodinated and brominated DNA have been made for phasing using MIR and MAD methods. This crystal structure and others with different DNA and HMG-protein sequences will reveal the molecular basis for sequence-tolerance of HMG-D, complementing ongoing thermodynamic studies, and answering specifically: (1) What is the interface of a non-sequence-specific protein-DNA complex like? (2) How differently do the non-sequence-specific and sequence-specific HMG-box proteins bind to DNA? (3) How does the HMG-box domain of HMG-D adapt to different DNA binding sites? Understanding the mode of binding and structural basis for sequence-tolerance of this family of chromosomal proteins may have implications for treatment of human disease. The non-sequence-specific HMG-domain proteins recognize cis-DDP lesions in vivo, and appear to be important in the function of a highly successful anti-tumor agent, cis-DDP. Structural studies with HMG-D will be valuable for understanding DNA recognition at a level of detail that will be helpful in the design of improved anti-tumor drugs.
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