Lorena S. Beese (Primary)
Chemical Biology, Computational Biology, Enzyme Mechanisms, Nucleic Acids Biochemistry, Protein Structure, Replication and Stabilization of Genes, Signal Transduction, Structural Biology, X-Ray Crystallography
Overview. Research in my laboratory is focused on understanding fundamental biological processes in atomic detail. A multi-disciplinary strategy is employed using macromolecular X-ray crystallography to determine high resolution, three-dimensional images of proteins and appropriate complexes. The structural information is combined with biochemical, biophysical, genetic, and computational analyses to address questions central to cancer biology. In addition to generating basic biological insights, this approach may facilitate the development new therapeutic agents for the treatment cancer and other diseases.
DNA Replication. My laboratory has contributed to understanding the molecular mechanisms that underlie accurate and mutagenic DNA replication. By integrating high-resolution X-ray structures with functional studies and computational analyses we have been able to elucidate key features that determine high-fidelity DNA replication. This work included exploitation of the properties of a DNA polymerase I that replicates DNA even when crystallized, allowing us to capture high-resolution snapshots of this polymerase in action. We are using this polymerase as a model system to study molecular mechanisms of DNA mis-pair incorporation and action of carcinogens that can lead to mutations. These approaches enabled us to (i) capture all mismatched bases bound in a polymerase active site and identified structural mechanisms that lead to polymerase stalling and subsequent error correction; (ii) establish the molecular basis for the mutagenicity of several classes of damaged DNA including O6-methyl-guanine lesions and 8-oxo guanine lesions; (iii) capture a tautomeric C-A mismatched base pair that is isosteric to a correct Watson-Crick base pair in the polymerase active site, providing the first direct evidence for the rare tautomer hypothesis for spontaneous mutagenesis noted by Watson and Crick in 1954.
Human DNA Mismatch Repair. We are applying the same approaches to the study of multi-protein DNA assemblies involved in human mismatch repair (MMR). Mismatch repair is essential for maintaining genomic stability of all organisms. Defects in genes involved in mismatch repair lead to elevated mutation rates, and confer a strong predisposition to tumorigenesis. In collaboration with Professor Paul Modrich, current experiments are focused on understanding the structures and molecular mechanisms of protein-DNA assemblies involved in the initiation of the human mismatch repair reaction. We have determined crystal structures and elucidated molecular mechanisms of DNA recognition and specificity for human mismatch repair proteins MSH2-MSH6 (MutSa), and extended these analyses to MSH2-MSH3 (MutSb). These proteins are large ABC-ATPases that are the primary sensors of DNA replication errors in MMR. This work contributes to understanding the etiology of cancer causing HNPCC mutations. My laboratory has determined the structure and catalytic mechanism of human exonuclease I and is working to understand how this nuclease is regulated in different DNA repair processes. Initially, identified in the context of MMR, this 5’ structure specific nuclease is involved in a number of DNA metabolic processes, including end resection in double strand break repair, telomere maintenance and rescuing stalled replication forks.
Post-translational lipid modifying enzymes. Numerous signal transduction proteins, including members of the Ras GTPase superfamily, require post-translational attachment of an isoprenoid lipid group for proper function. My laboratory has made fundamental discoveries elucidating the structure and molecular mechanisms of the human CaaX protein prenyltransferases that carry out this essential posttranslational modification. We determined the first structures of a protein farnesyltransferase (FTase), and protein geranylgeranyltransferase I (GGTase I). Subsequently, we have determined a series of structures representing the major steps along the reaction coordinate of these enzymes. From these observations can be deduced the structural determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes. These essential enzymes are considered promising targets both for the development of anticancer drugs and for treatment for infectious disease (see below). These studies have been extended to CaaX prenyltransferases from a number of human pathogens for the purpose of drug development (below).
Structure-based drug design. Inhibitors of FTase (FTIs) cause tumor regression in animals and are being evaluated in clinical trials for the treatment of human cancer and other human diseases. My laboratory has published a series of papers with chemists from both academia and industry that define binding modes for human farnesyltransferase inhibitors (FTIs) and describe their mechanism of inhibition. Additionally, we have collaborated with medicinal chemists on the structure-based development of both FTIs and GGTase-1 inhibitors (GGTIs) for the treatment of cancer and infectious diseases. My laboratory has contributed over fifty crystal structures of native FTase and GGTase-1 and mutant variants bound to substrates, products and inhibitors to the Protein Data Bank (PDB). We have extended these studies to prenyltranferases from human pathogens, most notably Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, and determined crystal structures, defined the enzymatic reaction cycle, determined the basis of ligand selection, and identified structurally divergent regions of the active site that can be exploited for development of specific inhibitors. We have identified novel anti-fungal compounds both by repurposing known human FTIs and through in silico screening of small molecule libraries. Several FTIs tested in vivo are fungicidal and are being optimized to improve potency.
Observing Enzymes in Action. A common theme of the laboratory is the study of enzyme mechanisms at near atomic resolution by determining three-dimensional structures that represent stages along the reaction pathway. Structural information is combined with biochemical, biophysical, and computational analyses to understand how enzymes function. An exciting direction arises from the observation that enzymes BF DNA polymerases I and human exonuclease I retain catalytic activity in the crystal. We are adopting a time-resolved X-ray crystallography strategy to the investigation of these enzyme mechanisms.
|1994-1997||Searle Scholar Award|
|2005||SER-CAT Outstanding Science Award|
|2005-2015||NIGMS (MERIT) Award|
|2009||Elected to National Academy of Sciences|
BA Oberlin College, Mathematics and Biology
PhD Brandeis University, Biophysics
Postdoctoral Fellow Yale University, Molecular Biophysics and Biochemistry Advisor: Thomas A. Steitz