Uracil-DNA glycosylase
Created by Erica Busch
Uracil-DNA glycosylase is important because it fixes errors in DNA, which stores genetic information for organisms. When non-native bases replace adenine, guanine, cytosine or thymine problems arise during replication and can lead to mutations and diseases.1 Uracil a RNA base can incorporate as a base pair to adenine, which will then mispair with guanine and be mutagenic. There are two ways that uracil can incorporate itself into DNA; DNA polymerase can misincorporate a deoxyuridine monophosphate (dUMP) resulting in an A-U pair or a cytosine that is already incorporated in DNA can spontaneously deaminate and result in a G-U mispair.1 Uracil DNA Glycolyase can initiate the Base Excision Repair pathway for Uracil by hydrolyzing the N-C1’ gylcosidic bond to result in a free uracil and an abasic site on the deoxyribose sugar.1
Uracil-DNA Glycosylase (UDG) from a Mycobacterium Tuberculosis (UDGmt), pdb id 2ZHX, has a molecular weight of 247075.14. UDGmt is composed of two unique sequences that repeat to make 14 chains. The first sequence makes up chain A and consists of 238 residues with 12 alpha helices, 4 beta sheets and 13 turns. It has a “single domain α/β fold, with a central, four-stranded parallel β-sheet surrounded by 8 α-helices.”1 The second sequence is for chain B and consists of 84 residues with 5 anti-parallel β-sheets surrounded by 2 amphipathic a-helices, and 15 turns.1 Chain A makes up the enzyme molecule of UDG, while Chain B is the inhibitor molecule. Chain B, the Uracil-DNA Glycosylase Inhibitor (Ugi), is a B. subtilis bacteriophage that inhibits UDG. Ugi mimics DNA as it would interact with UDG as Ugi closely resembles double stranded DNA.1,2
The structure of UDG’s isolated from human, viral and E. coli sources reveal 5 major structural patterns. They are: (1) the minor-grooveintercalation loop; (2) the Pro-Rich loop; (3) the Gly-Ser loop; (4) the uracil-specificity region; and (5) the water-activating loop.1
UDG undergoes a conformational change from ‘open’ to ‘closed’ by about 11°, when it binds to DNA at the C-terminal edge of UDG’s central β-sheet.1,3 UDG detects lesions by DNA backbone compression with Leu-272 that intercalates into the DNA base stack, slightly bending the DNA.1 The three rigid loops of UDG; the Pro-rich loop, the Gly-Ser loop and the minor-groove intercalation loop all contain serine, proline and glycine residues to facilitate a close approach of UDG to the DNA backbone.1 The flipped-out nucleotide is surrounded by DNA in the B-form that is compressed by 4Å because of the three rigid-loops. This causes the DNA to kink its helical axis by 2Å and bend by 45ᵒ.1 The damaged nucleotide is flipped out through the DNA major groove and into the enzyme’s active site, between β1 and β3.1,4 Once productive binding has occurred the Leu intercalation loop moves about 4Å to allow the Leucine to penetrate the DNA minor groove thus flipping uridine.4 Subsequently, the binding pocket that recognized the uracil nucleotide through complementary active-site residues pulls. Additionally Leu-272 works to stabilize the productive enzyme-substrate complex. When the active site closes upon substrate binding the uracil and deoxyribose in the flipped-out nucleotide is distorted, resulting in strain relieved by glycosidic bond cleavage.4
Ugi is the B chain of pdb id 2ZHX. The structure of the complex was determined by using molecular replacement and multiple isomorphous replacement methods.5 Ugi very closely resembles dsDNA, all negatively-charged residues orient and drive a long range of electrostatic associations with UDG and the regions of substrate-bound DNA that do not approach UDG are mimicked in Ugi as well.1 An important residue is Glu-20 which is the only negatively charged residue that replaces a phosphate interaction; it is important because it provides two adjacent hydrogen bond acceptors.1 Another important aspect of the bonding observed between Ugi and UDG is the Ugi Gln19 carbonyl flip, which is triggered by formation of the complex.6 Ugi is structurally between the kinked DNA observed in UDG:DNA and B-form DNA.6 Ugi inserts β1 into the active site groove and envelops the conserved UDG Leu-272 active site loop.5 The Leu-272 is completely exposed in the free enzyme and completely buried in the inhibitor-enzyme complex.5 Hydrophobic residues cluster from the Ugi β sheet and α2 of the protruding UDG loop, with the interface consisting of polar, charged residues, making 18 hydrogen bonds and 22 ordered bound waters to line the periphery.5 Ugi in complex with UDG forms a beta-zipper structure, named to indicate the reversible pairing between intramolecular beta-strands.6 The interface between Ugi and UDG is over one quarter of Ugi’s solvent-accessible surface and involves 22 Ugi residues and 22 UDG residues.5 Ugi has two ligand components, imidazole and sulfate ion
The binding site of UDGmt is unique from all other known structures of UDG in that it is rich in arginyl residues which provide increased electrostatic effects and also adds hydrogen bonds to make up for those lost due to changes in the amino acid sequence especially in the catalytic loop.3 The catalytic loop of UDGmt is also distinct from all other known UDGs. A citrate ion is found in the uracil-binding pocket.3 This citrate ion mimics the interactions that the protein exhibits when bound to uracil.3
One homolog is Uracil-DNA-Glycosylase from Deinococcus Radiodurans (UDGdr).5 This homolog is 247 residues and has a molecular weight of 27966.82. . Its PDB number is 2BOO A. Like chain A of my protein this homolog has 12 alpha helices as well, but it has 7 beta sheets, unlike the 4 that UDGmt has. UDGdr has a nitrate ligand associated whereas UDGmt has a citrate ion associated. UDGmt chain A and UDGdr have 43.4% sequence identity and overall 94% similarity.5 These two proteins are extremely structurally similar but vary in their amino acid sequence. They have a P value of 0, which measures the probability that the two structures are similar and a value of less than .05% indicates that they are very similiar.