Identification of a novel bifunctional uracil DNA glycosylase from Thermococcus barophilus Ch5

Abstract

Genomes of hyperthermophiles are facing a severe challenge due to increased deamination rates of cytosine induced by high-temperature, which could be counteracted by base excision repair mediated by uracil DNA glycosylase (UDG) or other repair pathways. Our previous work has shown that the two UDGs (Tba UDG247 and Tba UDG194) encoded by the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 can remove uracil from DNA at high temperature.Herein, we provide evidence that Tba UDG247 is a novel bifunctional glycosylase which can excise uracil from DNA and further cleave the phosphodiester bond of the generated apurinic/apyrimidinic (AP) site, which has never been described to date. In addition to cleaving uracil-containing DNA, Tba UDG247 can also cleave APcontaining ssDNA although at lower efficiency, thereby suggesting that the enzyme might be involved in the repair of AP site in DNA. Kinetic analyses showed that Tba UDG247 displays a faster rate for uracil excision than for AP cleavage, thus suggesting that cleaving AP site by the enzyme is a rate-limiting step for its bifunctionality. The phylogenetic analysis showed that Tba UDG247 is clustered on a separate branch distant from all the reported UDGs. Overall, we designated Tba UDG247 as the prototype of a novel family of bifunctional UDGs.

Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold

“…Previous work on DNA repair enzymes from Thermococcales reveals a variety of bifunctional glycosylases that remove damaged nucleobases and cleave the resulting AP sites. These enzymes, including AGOG ( 33 ), Tba uracil DNA glycosylase (UDG247) ( 34 ), endonuclease III ( 35 ) and Tb-Mig ( 36 ), encode both a primary repair glycosylase activity that first recognizes and removes damaged (or mismatched) nucleobases producing AP sites that are then cleaved by the AP lyase activity, with both activities on the same polypeptide. For example, the AGOG glycosylase activity cleaves 8-oxoG, and then the associated AP lyase activity cleaves the remaining AP site, leaving a one nucleotide gap with 3′ and 5′ phosphates ( 33 ).…”

Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS

“…Uracil-DNA glycosylase (UDG) is a key enzyme that cleaves the N-glycosidic bond thereby initiating the BER pathway. It has been shown that UDG genes are highly conserved across Streptomyces genus and that the deletion of these genes is not lethal in S. lividans (55). In the light of these results, it is conceivable that, in addition to UDGs, NucS Sam may play a significant role in the BER-like pathway in Streptomyces .…”