Dataset Information

Recognition of 1,N ²-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

ABSTRACT: The adenine, cytosine, and guanine bases of DNA are susceptible to alkylation by the aldehyde products of lipid peroxidation and by the metabolic byproducts of vinyl chloride pollutants. The resulting adducts spontaneously cyclize to form harmful etheno lesions. Cells employ a variety of DNA repair pathways to protect themselves from these pro-mutagenic modifications. Human alkyladenine DNA glycosylase (AAG) is thought to initiate base excision repair of both 1,N ⁶-ethenoadenine (?A) and 1,N ²-ethenoguanine (?G). However, it is not clear how AAG might accommodate ?G in an active site that is complementary to ?A. This prompted a thorough investigation of AAG-catalyzed excision of ?G from several relevant contexts. Using single-turnover and multiple-turnover kinetic analyses, we found that ?G in its natural ?G·C context is very poorly recognized relative to ?A·T. Bulged and mispaired ?G contexts, which can form during DNA replication, were similarly poor substrates for AAG. Furthermore, AAG could not recognize an ?G site in competition with excess undamaged DNA sites. Guided by previous structural studies, we hypothesized that Asn-169, a conserved residue in the AAG active-site pocket, contributes to discrimination against ?G. Consistent with this model, the N169S variant of AAG was 7-fold more active for excision of ?G compared with the wildtype (WT) enzyme. Taken together, these findings suggest that ?G is not a primary substrate of AAG, and that current models for etheno lesion repair in humans should be revised. We propose that other repair and tolerance mechanisms operate in the case of ?G lesions.

SUBMITTER: Thelen AZ

PROVIDER: S-EPMC7008384 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Recognition of 1,N2-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

Thelen Adam Z AZ O'Brien Patrick J PJ

The Journal of biological chemistry 20191227 6

The adenine, cytosine, and guanine bases of DNA are susceptible to alkylation by the aldehyde products of lipid peroxidation and by the metabolic byproducts of vinyl chloride pollutants. The resulting adducts spontaneously cyclize to form harmful etheno lesions. Cells employ a variety of DNA repair pathways to protect themselves from these pro-mutagenic modifications. Human alkyladenine DNA glycosylase (AAG) is thought to initiate base excision repair of both 1,N6-ethenoadenine ...[more]

PMID: 31882538

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Project description:Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (?A), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and ?A fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the ?A fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the ?A fluorescence. Although these mutations do not have large effects on the rate constant for excision of ?A, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs.

Project description:BackgroundThiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms.ResultsBased on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant.ConclusionsBased on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

Dataset Information

Recognition of 1,N ²-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

Publications

Recognition of 1,<i>N</i><sup>2</sup>-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

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Dataset Information

Recognition of 1,N 2-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

Publications

Recognition of 1,<i>N</i><sup>2</sup>-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Recognition of 1,N ²-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.