Our lab works on a variety of different biological systems including, DNA replication and repair, nuclear receptors, and heparan sulfate biosynthesis. Listed below are the highlights from this past year: 1) Human polymerase Mu (Pol Mu) is a Pol X family member involved in the repair of non-homologous DNA double strand breaks resulting from exogenous insults such as ionizing radiation or programmed DNA breaks during immunoglobulin kappa light chain rearrangement in VDJ recombination. Faithful repair of these breaks requires incorporation of a nucleotide containing the correct base, as well as sugar selectivity for deoxy- versus ribonucleotides. Incorporation of ribonucleotides can ultimately lead to genome instability due to hydrolytic backbone cleavage. Unlike most polymerases, including other Pol X family members Beta and Lambda, Pol Mu readily incorporates ribonucleotides in break repair. In collaboration with the Kunkel group, we reported in a manuscript (in press) to DNA Repair crystal structures of the catalytic cycle of ribonucleotide incorporation by Pol Mu. These structures reveal that Pol Mu incorporates ribonucleotides with no obvious distortions of active site geometry or DNA substrate when compared to deoxyribonucleotide incorporation. In addition, we were able to generate mutants that display moderately improved ribonucleotide discrimination in break repair. The results of this work suggest that no one single feature/residue of Pol Mu is responsible for its ability to incorporate ribonucleotides but rather the discrimination behaviors are a complex, synergistic system of numerous interactions within the active site microenvironment. 2) Glucocorticoids (GCs) are steroid hormones that regulate many essential physiological process such as metabolism and immune function through interactions with the Glucocorticoid Receptor (GR). Most GCs exert their effects through interaction with the GRalpha isoform of GR. GRbeta, a splice variant of GRalpha, displays dominate negative activity toward GRalpha. GRbeta differs from GRalpha by the replacement of the C-terminal 50 amino acids with a unique 15 amino acid sequence. In collaboration with the Cidlowski group, we solved the structure of the ligand binding domain of GRbeta to better understand its function. Our crystal structure reveals that the unique C-terminal tail of GRbeta is disordered when bound to its only known ligand, the GR antagonist RU-486. We were also able to demonstrate that GRbeta appears incapable of binding coactivator in the presence of RU-486 but can bind the corepressor. Follow up studies revealed that the C-terminal 15 amino acids are not required for ligand or corepressor binding. This study provides the molecular foundation for the development of drugs to inhibit the dominate negative activity of GRbeta on GRalpha. 3) Heparan Sulfates (HS) are polysaccharides involved in physiological and pathophysiological functions such as infection, inflammatory responses, blood coagulation, cancer, and embryonic development. In this study, we solved the structure of the 6-O-sulfotransferase in complex with a polysaccharide substrate to reveal the specificity requirements of this enzyme. We used this information to generate mutations that changed the specificity. This information is valuable to the technique of chemoenzymatic synthesis where these enzymes are being used to synthesize HS for drug development.
Showing the most recent 10 out of 55 publications
