DNA Repair

Deinococcus radiodurans (DR) and DNA Repair

Deinococcus radiodurans (DR) is a remarkable bacterium that is highly resistant to genotoxic chemicals, oxidative damage, high levels of ionizing and ultraviolet radiation, and dehydration. Amazingly, Deinococcus radiodurans can withstand radiation 3,000 times what it would take to kill a human. As such, it is of interest to DNA both for the bioremediation of mixed waste and as a model organism to understand mechanisms of DNA repair.

OBER is supporting a multiple PI project to study the structural biology of DNA repair in Deinococcus radiodurans (DR). This effort is led by Dr. Stephen Holbrook and collaborators, Dr. Ursula Schulze-Gahmen, Prof. David Wemmer, Prof. Steven Brenner, Prof. Sung-Hou Kim and Prof. James Berger, in the Physical Biosciences Division and Dr. Michael Kennedy from PNNL. This project integrates the techniques of X-ray crystallography (Holbrook, Schulze-Gahmen, Kim, Berger), NMR (Wemmer, Kennedy) and computational biology (Brenner) to optimize our understanding of protein structure and biological function.

Current research is concentrated on two sets of proteins: the Nudix protein family of nucleoside diphosphate hydrolases and proteins involved in recombinational DNA repair, especially the proteins of the RecFOR pathway. The researchers are examining the three-dimensional structures of these proteins by NMR and X-ray crystallography. Their goal is to understand the proteins' functions in DNA repair and the radiation resistance of DR and to explore the application of these proteins in organisms engineered for bioremediation.

The Nudix proteins are a diverse and ubiquitous family, found in all three kingdoms of life and from viruses to humans. The family has great sequence and functional diversity, but is thought to generally be involved in cellular housecleaning and homeostasis. The first Nudix protein characterized was MutT that hydrolyzes damaged nucleotide triphosphates to protect DNA from their incorporation. There are 21 members of this family in DR, twice that of any other organism, suggesting a role in the exceptional survivability of this bacterium. Drs. Holbrook, Kennedy, and Wemmer are studying this family of DR proteins.

Dr. Schulze-Gahmen is focusing on the RecFOR proteins that have not been structurally characterized in any organism. She has crystallized the RecO and RecR proteins as well as a complex of RecO with DNA. Crystallographic diffraction data on both the Nudix proteins and the RecFOR proteins is being collected at the Macromolecular Crystallography Facility of the LBNL Advanced Light Source synchrotron.
Prof. Steven Brenner is collecting and merging the DR structural data as well as sequence and structural information from other organisms into a theory of how Nudix proteins work and how their functions are related to the sequence.

DNA Repair and Genetic Evolution

In the Life Sciences Division, Dr. John Tainer's lab has made significant discoveries in DNA repair. The following is a synopsis of their research from their website: "Cells must balance DNA repair to preserve fidelity with DNA variation allowing evolutionary changes. As over 10,000 DNA bases per day are repaired in each human cell, DNA excision-repair enzymes are essential to cell survival and to protection against cancer-causing mutations. Surprisingly, DNA repair inhibitors may improve current radiation and chemotherapies for cancer by specifically killing cancer cells that unlike normal cells will often undergo DNA synthesis and cell division with unrepaired DNA resulting in their death. Our DNA repair enzyme structures show how damaged DNA bases are recognized and removed in atomic detail. These enzymes repair DNA by flipping the DNA nucleotides out from the double helix and into specific binding pockets that are ideal for the design of inhibitors for anti-cancer therapies. We confirmed our understanding of these binding pockets by deliberately altering the specificity of the DNA repair enzyme uracil-DNA glycosylase to remove cytosine or thymine from normal DNA resulting in mutator phenotypes in vivo. We found that the endonuclease III structure is representative of a superfamily of DNA repair enzymes and a key HhH motif that recognizes DNA backbone. Our new structure of the major DNA-repair APendonuclease, which cuts DNA at sites where bases are missing, defines its active site and identifies mechanism for recognizing missing bases."

The SIBYLS beamline at the ALS has been designed specifically to tackle the problems facing the crystallographic structure determinations of large complexes and to overcome experimental difficulties faced at existing X-ray sources. A major challenge in structural biology is to provide a detailed mechanistic understanding of the processes underlying the maintenance of genetic integrity. The emerging view from genetic, biochemical, and structural studies of DNA repair is that these activities are coordinately regulated by the assembly of large, dynamically-changing multi-protein complexes. Thus, the cellular roles of these repair activities cannot be completely rationalized through the biophysical chemistry of single protein components. The Structural Cell Biology (SCB) Core will directly target the multi-protein complexes that catalyze DNA repair, and it will serve as the structural cornerstone for the integration of the biochemical and structural studies that are at the heart of each of the projects within the SYBYLS program.

The Molecular Biology Consortium (THIS LINK DOESN'T WORK) at the ALS consists of academic institutions whose molecular biologists require macromolecular crystallographic information for their research. The intended purpose of MB-CAT is to build and operate synchrotron x-ray beamlines of the highest possible performance quality, to be used for molecular structural biology.