Sudip S. Parikh

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA.

Three high‐resolution crystal structures of DNA complexes with wild‐type and mutant human uracil‐DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage‐specific and damage‐general BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a ‘Ser–Pro pinch’ and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double‐stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate–sugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil‐containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP‐endonuclease, which catalyzes the first damage‐general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage‐specific and damage‐general steps of BER without requiring direct protein–protein interactions.

MutY catalytic core, mutant and bound adenine structures define specificity for DNA repair enzyme superfamily.

The DNA glycosylase MutY, which is a member of the Helix-hairpin-Helix (HhH) DNA glycosylase superfamily, excises adenine from mispairs with 8-oxoguanine and guanine. High-resolution crystal structures of the MutY catalytic core (cMutY), the complex with bound adenine, and designed mutants reveal the basis for adenine specificity and glycosyl bond cleavage chemistry. The two cMutY helical domains form a positively-charged groove with the adenine-specific pocket at their interface. The Watson-Crick hydrogen bond partners of the bound adenine are substituted by protein atoms, confirming a nucleotide flipping mechanism, and supporting a specific DNA binding orientation by MutY and structurally related DNA glycosylases.

Envisioning the molecular choreography of DNA base excision repair

Recent breakthroughs integrate individual DNA repair enzyme structures, biochemistry and biology to outline the structural cell biology of the DNA base excision repair pathways that are essential to genome integrity. Thus, we are starting to envision how the actions, movements, steps, partners and timing of DNA repair enzymes, which together define their molecular choreography, are elegantly controlled by both the nature of the DNA damage and the structural chemistry of the participating enzymes and the DNA double helix.

Lessons learned from structural results on uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) functions as a sentry guarding against uracil in DNA. UDG initiates DNA base excision repair (BER) by hydrolyzing the uracil base from the deoxyribose. As one of the best studied DNA glycosylases, a coherent and complete functional mechanism is emerging that combines structural and biochemical results. This functional mechanism addresses the detection of uracil bases within a vast excess of normal DNA, the features of the enzyme that drive catalysis, and coordination of UDG with later steps of BER while preventing the release of toxic intermediates. Many of the solutions that UDG has evolved to overcome the challenges of policing the genome are shared by other DNA glycosylases and DNA repair enzymes, and thus appear to be general.

Base excision repair enzyme family portrait: integrating the structure and chemistry of an entire DNA repair pathway

DNA base excision repair (BER) is essential to preserving the integrity of the genome. Recent crystallographic studies of representatives from each enzyme class required for BER reveal clues to the structural basis of an entire DNA repair pathway.

DNA damage recognition and repair pathway coordination revealed by the structural biochemistry of DNA repair enzymes

Cells have evolved distinct mechanisms for both preventing and removing mutagenic and lethal DNA damage. Structural and biochemical characterization of key enzymes that function in DNA repair pathways are illuminating the biological and chemical mechanisms that govern initial lesion detection, recognition, and excision repair of damaged DNA. These results are beginning to reveal a higher level of DNA repair coordination that ensures the faithful repair of damaged DNA. Enzyme-induced DNA distortions allow for the specific recognition of distinct extrahelical lesions, as well as tight binding to cleaved products, which has implications for the ordered transfer of unstable DNA repair intermediates between enzymes during base excision repair.

Structural Phylogenetics of DNA Base Excision Repair

Maintaining the chemical and informational integrity of DNA is vital for all cells. DNA both codes for the proteins and RNA essential for cellular metabolism, and provides the blueprints through which these instructions are passed to successive generations. As a hedge against devolution, a means of protecting the essential information inherent in primordial DNA must have arisen very early, and perhaps was mediated by recombination. The repair of DNA damage is fundamental to living organisms and is implicated for reactive oxygen and pathogen defenses, for controlling degenerative diseases and aging (Halliwell and Aruoma 1993), and for the development of sex and meiosis (Bernstein and Bernstein 1991).