Mutational Analysis of Arginine 276 in the Leucine-loop of Human Uracil-DNA Glycosylase

Abstract

Uracil residues are eliminated from cellular DNA by uracil-DNA glycosylase, which cleaves the N-glycosylic bond between the uracil base and deoxyribose to initiate the uracil-DNA base excision repair pathway. Co-crystal structures of the core catalytic domain of human uracil-DNA glycosylase in complex with uracil-containing DNA suggested that arginine 276 in the highly conserved leucine intercalation loop may be important to enzyme interactions with DNA. To investigate further the role of Arg 276 in enzyme-DNA interactions, PCR-based codonspecific random mutagenesis, and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg 276. All of the R276X mutant proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein in vitro, indicating that the active site structure of the mutant enzymes was not perturbed. The catalytic activity of the R276X preparations was reduced; the least active mutant, R276E, exhibited 0.6% of wildtype activity, whereas the most active mutant, R276H, exhibited 43%. Equilibrium binding studies utilizing a 2-aminopurine deoxypseudouridine DNA substrate showed that all R276X mutants displayed greatly reduced base flipping/DNA binding. However, the efficiency of UVcatalyzed cross-linking of the R276X mutants to singlestranded DNA was much less compromised. Using a concatemeric [ 32 P]U⅐A DNA polynucleotide substrate to assess enzyme processivity, human uracil-DNA glycosylase was shown to use a processive search mechanism to locate successive uracil residues, and Arg 276 mutations did not alter this attribute.

New Insights on the Role of the γ-Herpesvirus Uracil-DNA Glycosylase Leucine Loop Revealed by the Structure of the Epstein-Barr Virus Enzyme in Complex with an Inhibitor Protein

“…For human UNG the importance of residues located in this part of the loop had been shown by the drastic reduction of the catalytic efficiency for the excision of uracil bases in the context of double-stranded DNA when Arg276 is mutated 38 while activity against singlestranded DNA was less affected. A comparison of the sequences of the herpesvirus UNG leucine loops (Figure 1(b)) shows, that γ-herpesviruses share this loop structure (Figure 3(b)).…”

“…38 Whereas the first two functions are located on the part of the loop that is structurally conserved (sequence HPSPL in EBV), the last function is located on the more variable C-terminal side of the loop. Here the enzymes from HSV-1, D. radiodurans and Figure 1(a).…”

“…The importance of Arg276 for the enzyme activity towards uracil in doublestranded DNA has been shown by mutagenesis. 38 Whereas the human enzyme carries a tyrosine and an arginine residue, the tyrosine residue is replaced by a histidine in the E. coli and G. morhua enzymes, which conserve the same conformation of the loop (not shown). For D. radiodurans and HSV-1, the conformation of the loop differs due to the deletion of a residue (Figures 1(a) and 3(b)).…”

Reduced Hydrophobicity of the Minor Groove Intercalation Loop is Critical for Efficient Catalysis by Cold Adapted Uracil-DNA N-Glycosylase from Atlantic Cod

“…One explanation for this could be that the His275 residue binds stronger to the transition state than to the ground state, affecting k cat instead of K M [39]. A strong interaction between the leucine-loop in cUNG and DNA is supported by a mutational study of Arg276 in hUNG, which shows its importance for the activity of hUNG due to favourable interactions with DNA [40].…”

Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase

“…These observations suggest that Arg276 may stabilize the leucine-loop and Leu272 side chain either before or after it is inserted into the DNA minor groove. Recent mutational analysis of Arg276 demonstrated that substitutions at Arg276 resulted in reduction of steady-state uracil-DNA binding, base flipping, and enzyme catalysis [12].…”