Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis

Gehring, Alexandra M.; Zatopek, Kelly M; Burkhart, Brett W.; Потапов, В. А.; Santangelo, Thomas J.; Gardner, Andrew F.

doi:10.1016/j.dnarep.2019.102767

Cited by 11 publications

(17 citation statements)

References 74 publications

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“…Unlike for hOGG1, the DNA glycosylase activity of Pab-AGOG and Tga-AGOG appears to be only slightly affected by the base opposite GO in STO condition (lanes 2–5; Supplementary Figure S8 ) as it has been already observed for OGG2 ( 51 ). These results are in agreement with previous studies with Pae-AGOG and Tko-AGOG ( 19 , 23 ). For the concerted DNA glycosylase/AP lyase activity, similar results were observed for Pab-AGOG ( Supplementary Figure S8 , lanes 6 to 10), while Tga-AGOG displays a marked preference for a DNA substrate containing GO opposite a pyrimidine.…”

Section: Resultssupporting

confidence: 94%

“…The AGOG enzymes that have been most studied to date include those from Pyrobaculum aerophilum (Pae-AGOG) ( 19 , 20 ), Thermococcus gammatolerans (Tga-AGOG) ( 21 , 22 ) and Thermococcus kodakarensis (Tko-AGOG) ( 23 ) archaea, all of which live at around 90–100°C. The crystal structure of Pae-AGOG in its free and 8-oxoguanosine (8-oxodG)-bound forms reveal a significant difference from human OGG1/2 in terms of the structure of the HhH-GPD motif and the mode of GO recognition ( 20 ).…”

Section: Introductionmentioning

confidence: 99%

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Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing

Coste

Goffinont

Cros

et al. 2022

Nucleic Acids Research

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8-Oxoguanine (GO) is a major purine oxidation product in DNA. Because of its highly mutagenic properties, GO absolutely must be eliminated from DNA. To do this, aerobic and anaerobic organisms from the three kingdoms of life have evolved repair mechanisms to prevent its deleterious effect on genetic integrity. The major way to remove GO is the base excision repair pathway, usually initiated by a GO-DNA glycosylase. First identified in bacteria (Fpg) and eukaryotes (OGG1), GO-DNA glycosylases were more recently identified in archaea (OGG2 and AGOG). AGOG is the less documented enzyme and its mode of damage recognition and removing remains to be clarified at the molecular and atomic levels. This study presents a complete structural characterisation of apo AGOGs from Pyrococcus abyssi (Pab) and Thermococcus gammatolerans (Tga) and the first structure of Pab-AGOG bound to lesion-containing single- or double-stranded DNA. By combining X-ray structure analysis, site directed mutagenesis and biochemistry experiments, we identified key amino acid residues of AGOGs responsible for the specific recognition of the lesion and the base opposite the lesion and for catalysis. Moreover, a unique binding mode of GO, involving double base flipping, never observed for any other DNA glycosylases, is revealed. In addition to unravelling the properties of AGOGs, our study, through comparative biochemical and structural analysis, offers new insights into the evolutionary plasticity of DNA glycosylases across all three kingdoms of life.

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Section: Resultssupporting

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing

Coste

Goffinont

Cros

et al. 2022

Nucleic Acids Research

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“…BER is ubiquitous in bacteria, eukarya and archaea. Although it has extensively been studied in bacteria and eukarya, BER has thus far only been investigated in three archaea: the aerobic crenarchaeon P. aerophilum (Sartori et al 2001), the anaerobic euryarchaeon Archaeoglobus fulgidus (Knaevelsrud et al 2010) and the anaerobic euryarchaeon Thermococcus kodakarensis (Gehring et al 2020). Here, we present the first evidence that Tba UDG247 is able to remove uracil from DNA and further cleave the phosphodiester bond of the generated AP site, and may thus participate actively in the BER pathway in T. barophilus.…”

Section: Discussionmentioning

confidence: 88%

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

Zhang

Jiang

Gan

et al. 2021

Appl Microbiol Biotechnol

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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.

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“…TK0353 would not easily be identified by bioinformatic analysis as a DNA repair enzyme because it was annotated as a hypothetical protein and diverged from known DNA repair proteins. Because DNA damage occurs often in high-temperature environments, TK0353 may play a backup role in processing AP sites to complement the essential AP lyase in T. kodakarensis , endonuclease IV (TkoEndoIV) ( 27 ). Future studies will characterize TK0353 biochemistry in molecular detail.…”

Section: Discussionmentioning

confidence: 99%

Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes

Zatopek

Fossa

Bilotti

et al. 2022

Appl Environ Microbiol

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It has been predicted that 30-80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we have developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis ( T. kodakarensis ). Large DNA insert fosmid libraries representing a ∼5-fold average coverage of the T. kodakarensis genome were prepared in E. coli . RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing and capillary electrophoresis. The method described here provides details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next generation sequencing and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

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Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis

Cited by 11 publications

References 74 publications

Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing

Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing

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

Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes

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