The Crystal Structure of Mismatch-specific Uracil-DNA Glycosylase (MUG) from Deinococcus radiodurans Reveals a Novel Catalytic Residue and Broad Substrate Specificity

Elin Moe,Ingar Leiros,Arne O Smalås,Sean Mcsweeney

doi:10.1074/jbc.m508032200

Abstract

Deinococcus radiodurans is extremely resistant to the effects of ionizing radiation. The source of the radiation resistance is not known, but an expansion of specific protein families related to stress response and damage control has been observed. DNA repair enzymes are among the expanded protein families in D. radiodurans, and genes encoding five different uracil-DNA glycosylases are identified in the genome. Here we report the three-dimensional structure of the mismatch-specific uracil-DNA glycosylase (MUG) from D. radiodurans (drMUG) to a resolution of 1.75 angstroms. Structural analyses suggest that drMUG possesses a novel catalytic residue, Asp-93. Activity measurements show that drMUG has a modified and broadened substrate specificity compared with Escherichia coli MUG. The importance of Asp-93 for activity was confirmed by structural analysis and abolished activity for the mutant drMUGD93A. Two other microorganisms, Bradyrhizobium japonicum and Rhodopseudomonas palustris, possess genes that encode MUGs with the highest sequence identity to drMUG among all of the bacterial MUGs examined. A phylogenetic analysis indicates that these three MUGs form a new MUG/thymidine-DNA glycosylase subfamily, here called the MUG2 family. We suggest that the novel catalytic residue (Asp-93) has evolved to provide drMUG with broad substrate specificity to increase the DNA repair repertoire of D. radiodurans.

Highlights

Thymine DNA-glycosylase (TDG) removes uracil and thymine from G:U and G:T mismatches in double-stranded DNA [8]
Mismatch-specific Uracil-DNA Glycosylase from D. radiodurans fore, it has not been verified that the Mismatch-specific uracil-DNA glycosylase (MUG)/TDG enzymes follow the stepwise dissociative reaction pathway that is commonly accepted for uracil-DNA N-glycosylase (UNG)
The structure of drMUG is overall the same as of E. coli MUG; specificity analysis shows that drMUG possesses a different substrate specificity compared with Escherichia coli MUG (ecMUG) and is able to remove uracil from both G:U and A:U mismatches in double-stranded DNA (dsDNA) as well as uracil in single-stranded DNA, whereas ecMUG only removes uracil from G:U mismatches

Summary

EXPERIMENTAL PROCEDURES

Cloning—The gene encoding the G:U mismatch uracil-DNA glycosylase from D. radiodurans, dr0715, was inserted into the pDEST14 expression vector using the Gateway௡ technology according to the manufacturer’s manual (Invitrogen). Reactions between protein and DNA were performed in 20 mM Tris/ HCl, pH 8.0, 0.1 mg/ml bovine serum albumin, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 pmol of DNA, 1 pmol of drMUG and drMUGD93A, and 1 unit of ecMUG (Nordic Biosite AB) in a 20-␮l reaction mixture for 1 h at 37 °C. Complete data sets (see Table 2) were collected to a resolution of 1.75 and 1.7 Å for the native and mutant drMUGs, respectively. These data allowed the solution of both crystal structures using molecular replacement techniques. The crystal structure of the drMUGD93A mutant was determined by molecular replacement with MOLREP [21] using the refined structure of drMUG as a search model with water molecules, acetate, and Asp-93 removed. For an overview of the refinement statistics, see Table 2

RESULTS AND DISCUSSION

DrMUG DrUNG

Number of unique reflections