The Scripps Research Institute
Department of Molecular Biology
MB-4, 10550 North Torrey Pines Road
La Jolla, California 92037
The Scripps Research Institute
Department of Molecular Biology
MB-4, 10550 North Torrey Pines Road
La Jolla, California 92037
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The structure of human uracil-DNA glycosylase bound to
uracil-containing double-stranded DNA
revealed for the first time the complex interactions between
a critical human DNA repair enzyme
and a damaged piece of DNA. The 2.9Å crystal structure
of the enzyme-DNA complex suggests
that the enzyme
flips the entire uracil nucleotide out of the DNA double helix.
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The crystal structure of human deoxyuridine triphosphate pyrophosphatase (dUTPase) determined in its uncomplexed state and bound to dUMP, dUDP and the substrate dUTP have been determined at atomic resolution. The homotrimeric enzyme contains three equivalent active sites that lie at the three subunit interfaces and comprise conserved amino acid residues from all three subunits. Specific uracil binding by dUTPase represents a new nucleotide-recognition motif with implications for the evolution of RNA-peptide interactions. The structures suggest an obvious way for peptides to become early coenzymes in an RNA world, perhaps playing a role in the transiton to the current protein-DNA world.
Abasic sites arise continuously in DNA due to spontaneous depurination and depyrimidation, removal of uracil and mutagen-damaged bases from DNA by glycosylases such as uracil-DNA glycosylase. The repair of these lesions is critical since they can result in mutagenesis or cell death if left unrepaired. Exonuclease III (EC 3.1.11.2) is the major apurinic/apyrimidinic (AP) endonuclease in Escherichia coli. Exonuclease III acts on double-stranded DNA containing an abasic site and cleaves the DNA phosphodiester backbone 5' of the AP-site, creating a free 3'-OH for DNA repair synthesis which restores the correct DNA base.
Exonuclease III has an alpha,beta protein fold with the active site of the enzyme located at the bottom of a cleft between the two beta-sheets at one end of the molecule. Conserved residues at the metal binding site and the active site of Exonuclease III suggest that a single metal ion facilitates a nucleophilic attack by a water molecule on the phosphodiester backbone of DNA. A single Mn2+ ion binding to exonuclease III is observed by soaking the native, metal-free crystals in solutions containing Mn2+ and also in the structure of the ternary complex of the enzyme with Mn²+ and dCMP
Uracil-DNA glycosylase (EC 3.2.2.3) is the first enzyme in the base-excision repair pathway for removal of uracil from DNA. The successive steps in this pathway involve the action of an apurinic/apyrimidinic endonuclease, a DNA deoxyribophosphodiesterase, a DNA polymerase and a DNA ligase. Deamination of cytosine to uracil creates a premutagenic U:G mismatch that, unless repaired before the next round of replication, will cause a GC->AT transition mutation.
The structural basis for the exquisite specificity of UDG for uracil and for the probable roles of these conserved active site residues are evident in the structure of UDG in complex with the inhibitor 6-aminouracil. For uracil to reach its binding pocket within the UDG active site groove, the base must somehow be ``flipped out'' from the duplex DNA helix. Interaction with an extra-helical DNA base has been observed in the crystal structure of HhaI (cytosine-5)-methyltransferase, and now for the first time in a critical DNA repair enzyme in the structure of UDG bound to uracil-containing double-stranded DNA (see above).
The Bacillus subtilis bacteriophages PBS1 and PBS2 are unique in that their genomic DNA naturally contains uracil in place of thymine. These phages contain a small 84 amino acid Uracil-DNA Glycosylase Inhibitor (Ugi) protein that binds tightly to, and inactivates, UDGs from a range of organisms spanning E. coli to man. The 1.9Å crystal structure of the human UDG:Ugi protein complex revealed the basis for Ugi's broad specificity and essentially irreversible binding. Ugi targets the DNA-binding region of UDG by enveloping the critical conserved Leucine-272 residue and, thus, acts as a protein mimic of the interactions UDG makes with DNA.