Uracil DNA glycosylase (UDG) activities in Bradyrhizobium diazoefficiens: characterization of a new class of UDG with broad substrate specificity

Abstract

Repair of uracils in DNA is initiated by uracil DNA glycosylases (UDGs). Family 1 UDGs (Ung) are the most efficient and ubiquitous proteins having an exquisite specificity for uracils in DNA. Ung are characterized by motifs A (GQDPY) and B (HPSPLS) sequences. We report a novel dimeric UDG, Blr0248 (BdiUng) from Bradyrhizobium diazoefficiens. Although BdiUng contains the motif A (GQDPA), it has low sequence identity to known UDGs. BdiUng prefers single stranded DNA and excises uracil, 5-hydroxymethyl-uracil or xanthine from it. BdiUng is impervious to inhibition by AP DNA, and Ugi protein that specifically inhibits family 1 UDGs. Crystal structure of BdiUng shows similarity with the family 4 UDGs in its overall fold but with family 1 UDGs in key active site residues. However, instead of a classical motif B, BdiUng has a uniquely extended protrusion explaining the lack of Ugi inhibition. Structural and mutational analyses of BdiUng have revealed the basis for the accommodation of diverse substrates into its substrate binding pocket. Phylogenetically, BdiUng belongs to a new UDG family. Bradyrhizobium diazoefficiens presents a unique scenario where the presence of at least four families of UDGs may compensate for the absence of an efficient family 1 homologue.

Replication Protein A Enhances Kinetics of Uracil DNA Glycosylase on ssDNA and Across DNA Junctions: Explored with a DNA Repair Complex Produced with SpyCatcher/SpyTag Ligation

“…The small ssDNA sections that become available when individual DNA binding domains of RPA locally dissociate may be accessed by other proteins such as UNG2 [55] . Consistent with this idea, the catalytic domain of UNG2 binds small ssDNA sections that are similar in size to sites occupied by individual DNA binding domains of RPA (4–6 nt) and with similar association rates [48,49,55,59,60] . We speculate that the relatively high turnover by the covalent complex on 31 nt ssDNA indicated that RPA−Spy−UNG2 could participate in a handoff of ssDNA from the RPA portion of the complex to its UNG2 portion.…”

“…[55] Consistent with this idea, the catalytic domain of UNG2 binds small ssDNA sections that are similar in size to sites occupied by individual DNA binding domains of RPA (4-6 nt) and with similar association rates. [48,49,55,59,60] We speculate that the relatively high turnover by the covalent complex on 31 nt ssDNA indicated that RPAÀ SpyÀ UNG2 could participate in a handoff of ssDNA from the RPA portion of the complex to its UNG2 portion. This would imply that the two proteins do not need to physically dissociate for the handoff to occur, and that the winged-helix domain of RPA2 is not strictly required for the handoff other than to serve as a means for RPA to spatially recruit UNG2 to the ssDNA substrate.…”

An unconventional family 1 uracil DNA glycosylase in Nitratifractor salsuginis

“…The concentrations of Nsa UNG versus Ugi are shown in molar ratio. (B) Gel mobility shift analysis of binding between Ugi and hUNG /Nsa UNG . After incubation of enzyme with/without Ugi, reaction products were separated on 15% native PAGE gel in Tris‐glycine buffer ( pH 8.5).…”

Species-Specific Interactions of Arr with RplK Mediate Stringent Response in Bacteria

“…The arr gene was PCR amplified from M. smegmatis genomic DNA using the primers arr_NdeI_fp and arr_HindIII_rp and Pfu DNA polymerase. The amplicon of 440 bp obtained was gel purified and ligated to pTrcNdeIHis vector (41) to generate pTrcNdeIHis-arr. Further, pTrcNdeIHis-arr was digested with NcoI and HindIII to release arr fragment and subcloned into pACDH plasmid at the same restriction site to generate The PCR conditions were the following: initial denaturation at 94°C for 5 min and 94°C for 1 min, annealing at a gradient of 52°C to 60°C, extension at 70°C for 11 min, and a final extension at 70°C for 10 min.…”