We are studying structures and mechanisms of enzymes that repair chemical modifications of DNA in order to understand the molecular basis for DNA substrate selection and to identify the physical interactions that coordinate multistep repair processes. This work is being done as part a large collaborative project, "The Structural Cell Biology of DNA Repair," based at Lawrence Berkeley National Laboratory.
DNA excision repair proteins recognize chemically modified nucleotides and catalyze their removal from DNA. We are investigating the mechanisms of base-flipping and catalysis by DNA glycosylases through biochemical assays and crystallographic structure determinations. Our goal is to understand how these enzymes select damaged nucleotides in a vast excess of normal DNA. Bulky chemical adducts are excised from DNA by a large complex of nucleases and DNA remodeling enzymes formed during nucleotide excision repair (NER). We are investigating how the physical interactions of NER proteins specify the incision of the DNA backbone on either side of the damage.
The proteins encoded by the Fanconi Anemia damage response pathway constitute a multiprotein nuclear complex that is believed to function as a ubiquitin ligase. Activation of the Fanconi Anemia pathway results in monoubiquitination of the FancD2 protein and its co-localization to chromatin with other damage repair/response proteins. Biochemical and crystallographic studies done in collaboration with Alan D'Andrea are aimed at understanding the biological functions of the Fanconi Anemia proteins in signaling DNA damage, activating cell cycle checkpoints, and initiating the repair of DNA.
Mammals encode three DNA ligases that function in DNA replication, repair, or recombination pathways. The crystal structure of human DNA ligase in complex with a nicked DNA substrate (at right) showed that the enzyme encircles DNA, stabilizing a highly distorted conformation that exposes the DNA ends to the active site. DNA ligase I functions in the repair of Okazaki fragments in complex with PCNA, a ring-shaped processivity factor for replicative DNA polymerases. In collaboration with Alan Tomkinson, we are investigating the structural and functional consequences of this protein-protein interaction by a variety of approaches including small angle x-ray scattering at the SIBYLS beamline (Advanced Light Source, Lawrence Berkeley National Laboratory). Enzymological and crystallographic studies of human DNA ligase III (DNA repair) and ligase IV (nonhomologous DNA end joining) are also underway with the goal of understanding the mechanism(s) of DNA "nick-sensing."
Enzymes that assemble at the replication fork catalyze DNA unwinding and the coordinated synthesis of two DNA strands. We are using EM and x-ray crystallographic methods to determine the architecture of the bacteriophage T7 replisome. Crystallographic studies and small angle x-ray scattering experiments are being used to characterize the dynamic structures of human multisubunit DNA polymerases and the enzymes that repair Okazaki fragments following replication. We have a long-standing interest in the mechanism of templated DNA synthesis by DNA polymerases.
We are investigating bacteriophage T7 DNA replication complexes by x-ray crystallographic and biochemical methods to understand how DNA unwinding and templated synthesis of 2 DNA strands are coordinated at the replication fork. We are currently investigating the complex of primase-helicase and DNA polymerase proteins that initiates synthesis of Okazaki fragments on the lagging strand of the replication fork. Efforts are being made towards the crystallization of multisubunit mammalian DNA polymerases.
The site-specific DNA recombination reaction catalyzed by bacteriophage lambda integrase is a model system for studying the regulated exchange of DNA strands, with attractive features for the genetic engineering of mammalian cells. Lambda recombination is responsive to the changing physiology of the E. coli host, either inserting the phage genome into the E. coli chromosome or excising it. The DNA substrates for the integration and excision reactions are shaped differently by DNA bending proteins that loop the DNA around the integrase tetramer. We are determining crystal structures of higher order lambda recombination complexes to understand how the physical interactions of proteins and DNAs allosterically regulate the exchange of DNA strands.
The genome of Saccharomyces cerevisiae is divided into actively transcribed regions that are established by the assembly of Silent information regulator (Sir) proteins on chromatin. DNA silencing is an important means of controlling gene. DNA silencing is an important means of controlling gene expression and suppressing aberrant DNA recombination. The yeast Sir2, Sir3 and Sir4 proteins interact with one another, and with transcription factors and deacetylated histone tails in order to spread along chromosomes and form silent DNA. We are characterizing the assembly of Sir protein complexes by protein interaction assays and x-ray crystallographic studies of the protein complexes.
The assembly of transcriptionally silent DNA in budding yeast requires histone deacetylation by the silent information regulator Sir2. In collaboration with Danesh Moazed, we have recently determined a crystal structure of yeast Sir2 that reveals a key intermediate of the NAD-dependent deacetylation reaction and suggests a means of regulating enzymatic activity. The human Sir2 homologs, Sirtuins, are critical regulators of lifespan and contribute to the regulation of many essential cell precesses including tumor suppression, fat mobilization, insulin regulation, aging and DNA repair. We are using a multidisciplinary approach in collaboration with Shin Imai to understand how Sirtuins achieve biological specialization.
Small organic molecules have been invaluable as research tools for investigating biological systems, and as therapeutic agents. A high throughput screening facility for the identification of small molecules in the Department of Biochemistry & Molecular Biophysics at Washington University is working closely with our resarch group to identify small molecule ligaqnds that perturb enzymatic activities and protein-protein interactions, in vitro and in living cells. My research group is interested in small molecule ligands that perturb enzymatic activities and protein-protein interactions, in vitro and in living cells. Unlike traditional genetic methods, small molecules can selectively interfere with, or augment, a subset of protein functions in a reversible manner.
We are developing specific inhibitors of genetically-engineered DNA ligases in order to identify their separate or overlapping functions in replication, repair, and recombination processes. The large protein complexes formed by the Fanconi Anemia proteins are also targets for the development of small molecule ligands that selectively interfere with protein-protein interactions formed transiently during cellular responses to DNA damage. We have also been using this approach to understand the cellular functions of protein inhibitors of the serine/threonine phosphatase, calcineurin.
