Uracil DNA Glycosylase Superfamily Research Articles

Structural and functional coupling in cross-linking uracil-DNA glycosylase UDGX.

Enzymes in uracil-DNA glycosylase (UDG) superfamily are involved in removal of deaminated nucleobases such as uracil, methylcytosine derivatives such as formylcytosine and carboxylcytosine, and other base damage in DNA repair. UDGX is the latest addition of a new class to the UDG superfamily with a sporadic distribution in bacteria. UDGX type enzymes have a distinct biochemical property of cross-linking itself to the resulting AP site after uracil removal. Built on previous biochemical and structural analyses, this work comprehensively investigated the kinetic and enzymatic properties of Mycobacterium smegmatis UDGX. Kinetics and mutational analyses, coupled with structural information, defined the roles of E52, D56, D59, F65 of motif 1, H178 of motif 2 and N91, K94, R107 and H109 of motif 3 play in uracil excision and cross-linking. More importantly, a series of quantitative analyses underscored the structural coupling through inter-motif and intra-motif interactions and subsequent functional coupling of the uracil excision and cross-linking reactions. A catalytic model is proposed, which underlies this catalytic feature unique to UDGX type enzymes. This study offers new insight on the catalytic mechanism of UDGX and provides a unique example of enzyme evolution.

Screening of glycosylase activity on oxidative derivatives of methylcytosine: Pedobacter heparinus SMUG2 as a formylcytosine- and carboxylcytosine-DNA glycosylase

5-Methylcytosine (mC) is an epigenetic mark that impacts transcription, development, diseases including cancer and aging. The demethylation process involves Tet-mediated stepwise oxidation of mC to hmC, fC, or caC, excision of fC or caC by thymine-DNA glycosylase (TDG), and subsequent base excision repair. Thymine-DNA glycosylase (TDG) belongs to uracil-DNA glycosylase (UDG) superfamily, which is a group of enzymes that are initially found to be responsible for excising the deaminated bases from DNA and generating apurinic/apyrimidinic (AP) sites. mC oxidative derivatives may also be generated from Fenton chemistry and γ-irradiation. In screening DNA glycosylase activity in UDG superfamily, we identified new activity on fC- and caC-containing DNA in family 2 MUG/TDG and family 6 HDG enzymes. Surprisingly, we found a glycosylase SMUG2 from bacterium Pedobacter heparinus (Phe), a subfamily of family 3 SMUG1 DNA glycosylase, displayed catalytic activity towards not only DNA containing uracil, but also fC and caC. Given the sequence and structural differences between the family 3 and other family enzymes, we investigated the catalytic mechanism using mutational, enzyme kinetics and molecular modeling approaches. Mutational analysis and kinetics measurements identified I62, N63 and F76 of motif 1, and H205 of motif 2 in Phe SMUG2 as important catalytic residues, of which H205 of motif 2 played a critical role in catalyzing the removal of fC and caC. A catalytic model underlying the roles of these residues was proposed. The structural and catalytic differences between Phe SMUG2 and human TDG were compared by molecular modeling and molecular dynamics simulations. This study expands our understanding of DNA glycosylase capacity in UDG superfamily and provides insights into the molecular mechanism of fC and caC excision in Phe SMUG2.

Kinetic Analysis of a Suicidal DNA‐crosslinking Uracil DNA Glycosylase in UDG Superfamily

Uracil DNA glycosylase (UDG) is an enzyme to initiate base excision repair (BER) of DNA by removing the damaged base from the strand and forming an apurinic/apyrimidinic site (AP site). There are at least six families of enzymes found in UDG superfamily. All the UDGs contain three motifs in their catalytic centers, and the sequences of the motifs are conserved, guaranteeing the glycosylase activity. UDGX was a newly discovered protein of the UDG superfamily from Mycobacterium smegmatis, which was confirmed to be closer to the Family 4 UDGs (UDGa) than the other members by studying its structure and sequence. However, different with the Family 4 UDGs, an obvious loop was inserted in the motif 3 of UDGX. In addition, UDGX was the first reported UDG which can covalently crosslink to the AP site formed by base excision via its His109 on the inserted loop. Several mutants were generated by substituting the key residues in the motif 1 and motif 3 of the MsmUDGX. Uracil DNA glycosylase activity assays as well as uracil DNA crosslinking activity assays were performed with these mutants. It was observed that the E52A and D56A mutants retained both the uracil DNA glycosylase and DNA crosslinking activities, however the H109A, H109N and H109Q only retained the uracil DNA glycosylase activity but lost the DNA crosslinking activity due to the substitution of the His109. Moreover, the loss of both activities of the D59A and R107A mutants were observed. Followed by the activity assays, kinetic analyses were performed on the mutants and the UDGX wild type. The k2/Km of the wild type MsmUDGX on the G/U substrate was determined to be 1.8 ± 0.2 × 104 s-1M-1, which was 10-fold lower than the k2/Km of the Family 4 UDG (TthUDGa) reported. Compared to the wild type, E52A, H109A, H109N and H109Q mutants exhibited a significant decrease of the catalytic reaction rate, suggesting that not only the key residue Glu52 in the motif 1 but His109 may also play an important role in the uracil excision process. The Asp56 and Asp59 in the motif 1 were confirmed to form salt bridges with the Arg107 on the inserted loop in the motif 3, which were hypothesized to stabilize the loop during the interaction between the enzyme and the DNA substrates. Consistent with the hypothesis, the mutations on Asp59 and Arg107 caused the loss of both activities, but the D56A mutant retained both activities, and the kinetic analysis indicated only slight decrease of the reaction rate, which may suggest the Asp56 was not the key residue to stabilize the loop in the salt bridges. In conclusion, the kinetic analysis provides a quantitative assessment on the roles of key catalytic residues on uracil excision activity and DNA-crosslinking activity.

Nucleosomes and the three glycosylases: High, medium, and low levels of excision by the uracil DNA glycosylase superfamily

Human cells express the UDG superfamily of glycosylases, which excise uracil (U) from the genome. The three members of this structural superfamily are uracil DNA glycosylase (UNG/UDG), single-strand selective monofunctional uracil DNA glycosylase (SMUG1), and thymine DNA glycosylase (TDG). We previously reported that UDG is efficient at removing U from DNA packaged into nucleosome core particles (NCP) and is minimally affected by the histone proteins when acting on an outward-facing U in the dyad region. In an effort to determine whether this high activity is a general property of the UDG superfamily of glycosylases, we compare the activity of UDG, SMUG1, and TDG on a U:G wobble base pair using NCP assembled from Xenopus laevis histones and the Widom 601 positioning sequence. We found that while UDG is highly active, SMUG1 is severely inhibited on NCP and this inhibition is independent of sequence context. Here we also provide the first report of TDG activity on an NCP, and found that TDG has an intermediate level of activity in excision of U and is severely inhibited in its excision of T. These results are discussed in the context of cellular roles for each of these enzymes.

The uracil‐DNA glycosylase homolog UdgX from M. smegmatis as a potential platform for in vitro protein evolution

A uracil‐DNA glycosylase (UDG) homolog from Mycobacterium smegmatis, UdgX, has been reported to lack uracil excision activity but instead binds uracil‐containing DNA tightly even under denaturing conditions, implying a covalent protein‐DNA adduct (Sang et al., “A unique uracil‐DNA binding protein of the uracil DNA glycosylase superfamily” NAR 43, 8452–63, 2015). To determine whether UdgX could be used as a fusion partner to tether a protein library to its encoding DNA as a platform for in vitro protein evolution, we expressed, purified, and characterized UdgX for its uracil‐binding activity. As expected, UdgX exhibits specific complex formation with uracil‐containing single stranded and double stranded DNA substrates by electrophoretic mobility shift assay (EMSA), but only in the absence of SDS. This implies that the UdgX‐DNA interaction is non‐covalent, contradicting the earlier report. Further work will be necessary to determine whether the noncovalent UdgX‐DNA interaction is sufficiently strong to stably tether a protein library to its encoding DNA.Support or Funding InformationNew England Biolabs Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function.

SMUG2 DNA glycosylase from Pedobacter heparinus as a new subfamily of the UDG superfamily

Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are essential to maintain genome integrity, in which the base excision repair (BER) pathway plays a major role in the removal of base damage. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for excising the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Using bioinformatics tools, we identified a family 3 SMUG1-like DNA glycoyslase from Pedobacter heparinus (named Phe SMUG2), which displays catalytic activities towards DNA containing uracil or hypoxanthine/xanthine. Phylogenetic analyses show that SMUG2 enzymes are closely related to family 3 SMUG1s but belong to a distinct branch of the family. The high-resolution crystal structure of the apoenzyme reveals that the general fold of Phe SMUG2 resembles SMUG1s, yet with several distinct local structural differences. Mutational studies, coupled with structural modeling, identified several important amino acid residues for glycosylase activity. Substitution of G65 with a tyrosine results in loss of all glycosylase activity. The crystal structure of the G65Y mutant suggests a potential misalignment at the active site due to the mutation. The relationship between the new subfamily and other families in the UDG superfamily is discussed. The present study provides new mechanistic insight into the molecular mechanism of the UDG superfamily.

Structural Basis of Substrate Specificity in Geobacter metallireducens SMUG1

Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are critical to maintain genome integrity, in which the base excision repair pathway plays an essential role. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for removing the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Geobacter metallireducens SMUG1 (GmeSMUG1) is an interesting family 3 enzyme in the UDG superfamily, with dual substrate specificities for DNA with uracil or xanthine. In contrast, the mutant G63P of GmeSMUG1 has exclusive activity for uracil, while N58D is inactive for both substrates, as we have reported previously. However, the structural bases for these substrate specificities are not well understood. In this study, we solved a series of crystal structures of WT and mutants of GmeSMUG1 at relatively high resolutions. These structures provide insight on the molecular mechanism of xanthine recognition for GmeSMUG1 and indicate that H210 plays a key role in xanthine recognition, which is in good agreement with the results of our EMSA and activity assays. More importantly, our mutant structures allow us to build models to rationalize our previous experimental observations of altered substrate activities of these mutants.

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes.

A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs.

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb-A111N can form bidentate hydrogen bonds with the N3 and O4 moieties of the uracil base. Genetic analysis indicates the gain of function for A/U base pairs allows the MUG-K68N mutant to remove uracil incorporated into the genome during DNA replication. The implications of this study in the origin of life are discussed.

Uracil-DNA glycosylases-structural and functional perspectives on an essential family of DNA repair enzymes.

Uracil-DNA glycosylases (UDGs) are evolutionarily conserved DNA repair enzymes that initiate the base excision repair pathway and remove uracil from DNA. The UDG superfamily is classified into six families based on their substrate specificity. This review focuses on the family I enzymes since these are the most extensively studied members of the superfamily. The structural basis for substrate specificity and base recognition as well as for DNA binding, nucleotide flipping and catalytic mechanism is discussed in detail. Other topics include the mechanism of lesion search and molecular mimicry through interaction with uracil-DNA glycosylase inhibitors. The latest studies and findings detailing structure and function in the UDG superfamily are presented.

Evolutionary pattern of four representative DNA repair proteins across six model organisms: an in silico analysis

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to DNA molecules that encodes its genome. In this study, we examined evolution of O6-Methylguanine DNA alkyltransferase (MGMT), Xeroderma pigmentosum group D (XPD) protein, G/T mismatch-specific DNA glycosylases, MutS DNA repair proteins of Escherichia coli, Pyrococcus kodakaraensis, Saccharomyces cerevisae, Drosophila melanogester, Mus musculus, and Homo sapiens, which are involved in direct repair, Nucleotide excision repair, base excision repair, and mismatch repair, respectively. Sequence and domain analysis of these proteins indicates that during the course of evolution catalytic residues in the catalytic domain remain conserved. Phylogenetic tree analysis suggested that MGMT proteins of human and mouse have archaeal origin, whereas XPD proteins which are responsible for nucleotide excision repair evolved progressively from lower organism to higher organism. G/T mismatch-specific DNA glycosylases belong to family 1, family 2, and family 4 of uracil-DNA glycosylases superfamily. Family 2 proteins have broader substrate specificity in comparison to proteins of other families. In eukaryotic organisms, prokaryotic MUTS genes are duplicated and various paralogs are present. No unified evolution mechanism can explain the evolution of all these DNA repair proteins. Large sequence variation is observed among same DNA repair proteins of different organisms. However, residues involved DNA repair work and DNA binding remain conserved during the course of evolution.

Specificity and Catalytic Mechanism in Family 5 Uracil DNA Glycosylase

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily.

New Family of Deamination Repair Enzymes in Uracil-DNA Glycosylase Superfamily

DNA glycosylases play a major role in the repair of deaminated DNA damage. Previous investigations identified five families within the uracil-DNA glycosylase (UDG) superfamily. All enzymes within the superfamily studied thus far exhibit uracil-DNA glycosylase activity. Here we identify a new class of DNA glycosylases in the UDG superfamily that lacks UDG activity. Instead, these enzymes act as hypoxanthine-DNA glycosylases in vitro and in vivo. Molecular modeling and structure-guided mutational analysis allowed us to identify a unique catalytic center in this class of DNA glycosylases. Based on unprecedented biochemical properties and phylogenetic analysis, we propose this new class of DNA repair glycosylases that exists in bacteria, archaea, and eukaryotes as family 6 and designate it as the hypoxanthine-DNA glycosylase family. This study demonstrates the structural evolvability that underlies substrate specificity and catalytic flexibility in the evolution of enzymatic function.

Phylogenomic Analysis of the Uracil-DNA Glycosylase Superfamily

The spontaneous deamination of cytosine produces uracil mispaired with guanine in DNA, which will produce a mutation, unless repaired. In all domains of life, uracil-DNA glycosylases (UDGs) are responsible for the elimination of uracil from DNA. Thus, UDGs contribute to the integrity of the genetic information and their loss results in mutator phenotypes. We are interested in understanding the role of UDG genes in the evolutionary variation of the rate and the spectrum of spontaneous mutations. To this end, we determined the presence or absence of the five main UDG families in more than 1,000 completely sequenced genomes and analyzed their patterns of gene loss and gain in eubacterial lineages. We observe nonindependent patterns of gene loss and gain between UDG families in Eubacteria, suggesting extensive functional overlap in an evolutionary timescale. Given that UDGs prevent transitions at G:C sites, we expected the loss of UDG genes to bias the mutational spectrum toward a lower equilibrium G + C content. To test this hypothesis, we used phylogenetically independent contrasts to compare the G + C content at intergenic and 4-fold redundant sites between lineages where UDG genes have been lost and their sister clades. None of the main UDG families present in Eubacteria was associated with a higher G + C content at intergenic or 4-fold redundant sites. We discuss the reasons of this negative result and report several features of the evolution of the UDG superfamily with implications for their functional study. uracil-DNA glycosylase, mutation rate evolution, mutational bias, GC content, DNA repair, mutator gene.

Identification of Escherichia coli Mismatch-specific Uracil DNA Glycosylase as a Robust Xanthine DNA Glycosylase

The gene for the mismatch-specific uracil DNA glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the human thymine DNA glycosylase with activity against mismatched uracil base pairs. Examination of cell extracts led us to detect a previously unknown xanthine DNA glycosylase (XDG) activity in E. coli. DNA glycosylase assays with purified enzymes indicated the novel XDG activity is attributable to MUG. Here, we report a biochemical characterization of xanthine DNA glycosylase activity in MUG. The wild type MUG possesses more robust activity against xanthine than uracil and is active against all xanthine-containing DNA (C/X, T/X, G/X, A/X and single-stranded X). Analysis of potentials of mean force indicates that the double-stranded xanthine base pairs have a relatively narrow energetic difference in base flipping, whereas the tendency for uracil base flipping follows the order of C/U > G/U > T/U > A/U. Site-directed mutagenesis performed on conserved motifs revealed that Asn-140 and Ser-23 are important determinants for XDG activity in E. coli MUG. Molecular modeling and molecular dynamics simulations reveal distinct hydrogen-bonding patterns in the active site of E. coli MUG that account for the specificity differences between E. coli MUG and human thymine DNA glycosylase as well as that between the wild type MUG and the Asn-140 and Ser-23 mutants. This study underscores the role of the favorable binding interactions in modulating the specificity of DNA glycosylases.

Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway

Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway.

Helix–hairpin–helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues

The mutagenic threat of hydrolytic DNA cytosine deamination is met mostly by uracil DNA glycosylases (UDG) initiating base excision repair. However, several sequenced genomes of archaeal organisms are devoid of genes coding for homologues of the otherwise ubiquitous UDG superfamily of proteins. Previously, two possible solutions to this problem were offered by (i) a report of a newly discovered family of uracil DNA glycosylases exemplified by MJ1434, a protein found in the hyperthermophilic archaeon Methanocaldococcus jannaschii, and (ii) the description of TTC0482, an EndoIV homologue from the hyperthermophilic bacterium Thermus thermophilus HB27, as being able to excise uracil from DNA. Sequence homologues of both proteins can be found throughout the archaeal domain of life. Three proteins orthologous to MJ1434 and the family founder itself were tested for but failed to exhibit DNA uracil glycosylase activity when produced in an Ung-deficient Escherichia coli host. Likewise, no DNA uracil processing activity could be detected to be associated with TTC0482, while the protein was fully active as an AP endonuclease. We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme. Use of Δung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard.

Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components

Hydrolytic deamination of DNA cytosine residues results in U/G mispairs, pre-mutagenic lesions threatening long-term genetic stability. Hence, DNA uracil repair is ubiquitous throughout all extant life forms and base excision repair, triggered by a uracil DNA glycosylase (UDG), is the mechanistic paradigm adopted, as it seems, by all bacteria and eukaryotes and a large fraction of archaea. However, members of the UDG superfamily of enzymes are absent from the extremely thermophilic archaeon Methanothermobacter thermautotrophicus ΔH. This organism, as a hitherto unique case, initiates repair by direct strand incision next to the DNA-U residue, a reaction catalyzed by the DNA uridine endonuclease Mth212, an ExoIII homologue. To elucidate the detailed mechanism, in particular to identify the molecular partners contributing to this repair process, we reconstituted DNA uracil repair in vitro from only four purified enzymes of M. thermautotrophicus ΔH. After incision at the 5′-side of a 2′-d-uridine residue by Mth212 DNA polymerase B (mthPolB) is able to take over the 3′-OH terminus and carry out repair synthesis generating a 5′-flap structure that is resolved by mthFEN, a 5′-flap endonuclease. Finally, DNA ligase seals the resulting nick. This defines mechanism and minimal enzymatic requirements of DNA-U repair in this organism.

Repair of deaminated base damage by Schizosaccharomyces pombe thymine DNA glycosylase

Thymine DNA glycosylases (TDG) in eukaryotic organisms are known for their double-stranded glycosylase activity on guanine/uracil (G/U) base pairs. Schizosaccharomyces pombe (Spo) TDG is a member of the MUG/TDG family that belongs to a uracil DNA glycosylase superfamily. This work investigates the DNA repair activity of Spo TDG on all four deaminated bases: xanthine (X) and oxanine (O) from guanine, hypoxanthine (I) from adenine, and uracil from cytosine. Unexpectedly, Spo TDG exhibits glycosylase activity on all deaminated bases in both double-stranded and single-stranded DNA in the descending order of X > I > U ≫ O. In comparison, human TDG only excises deaminated bases from G/U and, to a much lower extent, A/U and G/I base pairs. Amino acid substitutions in motifs 1 and 2 of Spo TDG show a significant impact on deaminated base repair activity. The overall mutational effects are characterized by a loss of glycosylase activity on oxanine in all five mutants. L157I in motif 1 and G288M in motif 2 retain xanthine DNA glycosylase (XDG) activity but reduce excision of hypoxanthine and uracil, in particular in C/I, single-stranded hypoxanthine (ss-I), A/U, and single-stranded uracil (ss-U). A proline substitution at I289 in motif 2 causes a significant reduction in XDG activity and a loss of activity on C/I, ss-I, A/U, C/U, G/U, and ss-U. S291G only retains reduced activity on T/I and G/I base pairs. S163A can still excise hypoxanthine and uracil in mismatched base pairs but loses XDG activity, making it the closest mutant, functionally, to human TDG. The relationship among amino acid substitutions, binding affinity and base recognition is discussed.