Molecular recognition in protein-DNA assemblies
Davey, Curt Alexander
Date of Issue2008
School of Biological Sciences
We initially completed our first project on designing and characterizing synthetic DNA-bending factors as tools for investigating and controlling gene regulation and DNA recombination. Engineered single chain proteins based on the bacterial integration host factor, scIHFs, hold promise for the design of a system for gene delivery utilizing the phage λ integrase site-specific DNA integration machinery. We discovered that one of the scIHF variants behaves as a divalent metal-mediated switch, controlling protein-induced bending and efficiency of DNA recombination. This holds potential for the design of metal-mediated switches for constructing DNA nano-devices and investigating functional relationships. We are also engaged in structural and functional studies of High Mobility Group A (HMGA) proteins. These small proto-oncoproteins are involved in an astonishing variety of nuclear processes, capable of regulating the expression of numerous genes and facilitating tumor development, metastasis, and viral progression. We found that multiple molecules of the HMGA2 subtype protein are able to bind to a single nucleosome core particle (NCP). We are presently working towards crystal structure characterization of DNA- and NCP-HMGA assemblies.Through structural analysis of a NCP containing a novel DNA construct, we discovered that even minute alterations in sequence can elicit dramatic structural changes in the DNA. This holds significance for genomic activities, like retroviral integration, that exploit extreme distortion of nucleosomal DNA, and provides additional evidence that nucleosomal DNA is rich in conformational character, which we can potentially take advantage of. Investigation of new DNA constructs may not only yield valuable sequence-versus-conformation relationships for the nucleosome, but would function in a synergistic capacity with our other projects, in particular for drug design and functional investigations, giving us a variety of substrates to work with. We more recently completed an in depth DNA footprinting and X-ray crystallographic study of platinum anticancer drug reaction with a NCP and the corresponding histone-free DNA. Distinctions in DNA and nucleosomal site selectivity were apparent between cisplatin and oxaliplatin, which could be linked to differences in their pharmacological profiles. Although DNA sequence seemed to dominate site preference, histone octamer association was capable of modulating adduct formation by both inhibiting or promoting platination at certain sites. Extensive adduct formation observed at the centre of the NCP suggests that this region of the nucleosome, known to be least accessible to protein factors, may provide a favorable target for the design of improved platinum anticancer drugs.In addition to platinum drug site selectivity studies, we engaged in biophysical and biochemical studies revealing functional aspects of chromatin-drug interactions that are shedding light on new possibilities for anticancer agent development. We showed that platinum adducts formed by reaction of cisplatin or oxaliplatin with the nucleosome core inhibit histone octamer-DNA sliding, but do not cause significant alteration of nucleosome positioning. Thus, adduct formation reinforces positional preferences intrinsic to the DNA sequence, which indicates that modulation of platinum drug site selectivity by histone octamer association may relate to nucleosome-specific properties of DNA. This sheds light on platinum drug-mediated inhibition of chromatin remodeling in vivo and suggests that adducts can shield their own repair and interfere with genomic activities by directly altering nucleosome dynamics.
DRNTU::Science::Biological sciences::Human anatomy and physiology::Deoxyribonucleic acids