DNA is the major information carrier in biological systems. The goal of this research program is to use x-ray diffraction to study changes in the three-dimensional structure of DNA since these changes may reflect different biological activities. The altered DNA conformations that are to be studied include left-handed Z-DNA, triple-stranded and quadruple-stranded DNA. When a segment of DNA goes from right handed to left handed, it is flanked by two B-Z junctions where the DNA goes from right handed to left handed.We are interested in studying the three-dimensional structure of that junction.Z-DNA can also be stabilized by the binding of peptides and proteins. We have crystallized a segment of Z-DNA bound to a lysine-rich peptide. Solution of its crystal structure may tell us about the detailed interactions that stabilize the molecule in the left-handed conformation. A number of proteins have been identified that bind specifically to Z-DNA. A major goal is to determine the structure of Z-DNA bound to one of these proteins. These include monoclonal antibodies or fragments thereof that are specific for Z-DNA. Antibodies of this type are found in the human disease systemic lupus erythematosis. Zuotin is a yeast protein that binds to Z-DNA specifically. The protein is being expressed with a view toward co-crystallization. A system has been developed for identifying segments of genes that flip into the left-handed Z form when the gene is actively transcribed. Experiments will be carried out on mammalian cells transfected with a Z- DNA binding protein to identify these Z-DNA forming segments and to determine the influence of Z-DNA binding proteins in the nucleus on cell transcriptional activities. Other transfection experiments will involve constructs using luciferase as a reporter gene. These plasmid constructs will have segments of the promoter region of c-myc, modified by site- directed mutagenesis. Those segments which have been identified as forming Z-DNA will have the sequences altered so that they will no longer form Z- DNA. The functional consequences of this will be determined. X-ray crystallographic studies will be carried out on triple-stranded DNA to understand the interactions between bases that stabilize triplexes.These will include homopurine-homopyrimidine triplexes as well as those formed by mixed sequence DNA.Triplexes have been found to be intermediates in the activity of recombination enzymes. DNA also can form four-stranded complexes. One of these involves four guanine residues hydrogen bonded together in a G-quartet Several crystals are available containing guanine quartets, and solution of their structure will enable us to understand those features which are important for stabilization, especially the role of ions. Another type of four-stranded structure is postulated to occur in cytosine-rich DNA sequences. It has been proposed that two duplexes held together by C.C+ interactions form a four-stranded complex by intercalation of base pairs from each duplex. We have several crystals of cytosine-rich sequences and hope to solve these in order to uncover the structural basis of the association of cytosine-rich strands.
