Base J, found in nuclear DNA of Trypanosoma brucei, is not a target for DNA glycosylases

Base excision repair (BER) is an evolutionarily conserved system which removes altered bases from DNA. The initial step in BER is carried out by DNA glycosylases which recognize altered bases and cut the N-glycosylic bond between the base and the DNA backbone. In kinetoplastid flagellates, such as Trypanosoma brucei, the modified base beta-D-glucosyl-hydroxymethyluracil (J) replaces a small percentage of thymine residues, predominantly in repetitive telomeric sequences. Base J is synthesized at the DNA level via the precursor 5-hydroxymethyluracil (5-HmU). We have investigated whether J in DNA can be recognized by DNA glycosylases from non-kinetoplastid origin, and whether the presence of J and 5-HmU in DNA has required modifications of the trypanosome BER system. We tested the ability of 15 different DNA glycosylases from various origins to excise J or 5-HmU paired to A from duplex oligonucleotides. No excision of J was found, but 5-HmU was excised by AlkA and Mug from Escherichia coli and by human SMUG1 and TDG, confirming previous reports. In a combination of database searches and biochemical assays we identified several DNA glycosylases in T. brucei, but in trypanosome extracts we detected no excision activity towards 5-HmU or ethenocytosine, a product of oxidative DNA damage and a substrate for Mug, TDG and SMUG1. Our results indicate that trypanosomes have a BER system similar to that of other organisms, but might be unable to excise certain forms of oxidatively damaged bases. The presence of J in DNA does not require a specific modification of the BER system, as this base is not recognized by any known DNA glycosylase.     

J-binding protein increases the level and retention of the unusual base J in trypanosome DNA

The nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base beta-d-glucosyl-hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences. We have described a 100 kDa protein that specifically binds to J in duplex DNA. We have now disrupted the genes for this J-binding protein (JBP) in T. brucei. The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences. Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild-type level of J in their DNA. Excess J, randomly introduced into T. brucei DNA by growing the cells in the presence of the J precursor 5-hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal. In contrast, cells containing JBP lose excess J only sluggishly. We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J. We propose that JBP is a novel DNA modification maintenance protein.     

Oxanine DNA glycosylase activities in mammalian systems

DNA bases carrying an exocyclic amino group, namely adenine (A), guanine (G) and cytosine (C), encounter deamination under nitrosative stress. Oxanine (O), derived from deamination of guanine, is a cytotoxic and potentially mutagenic lesion and studies of its enzymatic repair are limited. Previously, we reported that the murine alkyladenine glycosylase (Aag) acts as an oxanine DNA glycosylase (JBC (2004), 279: 38177). Here, we report our recent findings on additional oxanine DNA glycosylase (ODG) activities in Aag knockout mouse tissues and other mammalian tissues. Analysis of the partially purified proteins from the mammalian cell extracts indicated the existence of ODG enzymes in addition to Aag. Data obtained from oxanine DNA cleavage assays using purified human glycosylases demonstrated that two known glycosylases, hNEIL1 and hSMUG1, contained weak but detectable ODG activities. ODG activity was the highest in hAAG and lowest in hSMUG1.     

Mutational analysis of the damage-recognition and catalytic mechanism of human SMUG1 DNA glycosylase

Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through π–π stacking to the base (Phe98) and specific hydrogen bonds to the Watson–Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87–Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.     

Mammalian 5-formyluracil-DNA glycosylase. 2. Role of SMUG1 uracil-DNA glycosylase in repair of 5-formyluracil and other oxidized and deaminated base lesions

In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.     

Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes.

Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in eukaryotes and prokaryotes [1][2][3]. This enzyme removes uracil bases that are present in DNA as a result of either deamination of cytosine or misincorporation of dUMP instead of dTMP [4] [5], and it is the primary activity in the DNA base excision repair pathway. Although UDG activities have been shown to be present in several thermophiles [6][7][8], no sequences have been found that are complementary to the Escherichia coli ung gene, which encodes UDG [9]. Here, we describe a UDG from the thermophile Thermotoga maritima. The T. maritima UDG gene has a low level of homology to the E. coli G-T/U mismatch-specific DNA glycosylase gene (mug). The expressed protein is capable of removing uracil from DNA containing either a U-A or a U-G base pair and is heat-stable up to 75 degrees C. The enzyme is also active on single-stranded DNA containing uracil. Analogous genes appear to be present in several prokaryotic organisms, including thermophilic and mesophilic eubacteria as well as archaebacteria, the human-disease pathogens Treponema palladium and Rickettsia prowazekii, and the extremely radioresistant organism Deinococcus radiodurans. These findings suggest that the T. maritima UDG is a member of a new class of DNA repair enzymes.     

A novel DNA nucleotide in Trypanosoma brucei only present in the mammalian phase of the life-cycle.

The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with PstI and PvuII (Bernards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdJ and pdV, in T.brucei DNA, using the 32P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyldeoxyuridine 5'-monophosphate (pHOMedU). In contrast, nucleotide pdJ was exclusively present in bloodstream form trypanosomal DNA. Levels of pdJ were higher in DNA enriched for telomeric sequences than in total genomic DNA and pdJ was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be different from the known eukaryotic unusual DNA bases 5-methylcytosine, N6-methyladenine and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, alpha-putrescinylthymine, 5-dihydroxypentyluracil and N6-carbamoylmethyladenine. We conclude that pdJ is a novel eukaryotic DNA nucleotide and that it is probably responsible for the partial resistance to cleavage by PvuII and PstI of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.     

Dynamics of uracil and 5-fluorouracil in DNA

The prodrug 5-fluorouracil (5-FU), after activation into 5-F-dUMP, is an extensively used anticancer agent that inhibits thymidylate synthase and leads to increases in dUTP and 5-F-dUTP levels in cells. One mechanism for 5-FU action involves DNA polymerase mediated incorporation of dUTP and 5-F-dUTP into genomic DNA leading to U/A, 5-FU/A, or 5-FU/G base pairs. These uracil-containing lesions are recognized and excised by several human uracil excision repair glycosylases (hUNG2, hSMUG2, and hTDG) leading to toxic abasic sites in DNA that may precipitate cell death. Each of these enzymes uses an extrahelical base recognition mechanism, and previous studies with UNG have shown that extrahelical recognition is facilitated by destabilized base pairs possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of base pairs containing 5-FU and U are an important unknown in understanding the role of these enzymes in damage recognition and prodrug activation. The pH dependence of the (19)F NMR chemical shift of 5-FU imbedded in a model trinucleotide was used to obtain a pK(a) = 8.1 for its imino proton (10 °C). This is about 1.5 units lower than the imino protons of uracil or thymine and indicates that at neutral pH 5-FU exists significantly as an ionized tautomer that can mispair with guanine during DNA replication. NMR imino proton exchange measurements show that U/A and 5-FU/A base pairs open with rate constants (k(op)) that are 6- and 13-fold faster than a T/A base pair in the same sequence context. In contrast, these same base pairs have apparent opening equilibrium constants (αK(op)) that differ by less than a factor of 2, indicating that the closing rates (k(cl)) are enhanced by nearly equal amounts as k(op). These dynamic measurements are consistent with the previously proposed kinetic trapping model for extrahelical recognition by UNG. In this model, the enhanced intrinsic opening rates of destabilized base pairs allow the bound glycosylase to sample dynamic extrahelical excursions of thymidine and uracil bases as the first step in recognition.     

Telomeric co-localization of the modified base J and contingency genes in the protozoan parasite Trypanosoma cruzi

Base J or beta-d-glucosylhydroxymethyluracil is a modification of thymine residues within the genome of kinetoplastid parasites. In organisms known to contain the modified base, J is located mainly within the telomeric repeats. However, in Trypanosoma brucei, a small fraction of J is also located within the silent subtelomeric variant surface glycoprotein (VSG) gene expression sites, but not in the active expression site, suggesting a role for J in regulating telomeric genes involved in pathogenesis. With the identification of surface glycoprotein genes adjacent to telomeres in the South American Trypanosome, Trypanosoma cruzi, we became interested in the telomeric distribution of base J. Analysis of J and telomeric repeat sequences by J immunoblots and Southern blots following DNA digestion, reveals approximately 25% of J outside the telomeric repeat sequences. Moreover, the analysis of DNA sequences immunoprecipitated with J antiserum, localized J within subtelomeric regions rich in life-stage-specific surface glycoprotein genes involved in pathogenesis. Interestingly, the pattern of J within these regions is developmentally regulated. These studies provide a framework to characterize the role of base J in the regulation of telomeric gene expression/diversity in T. cruzi.     

Modulation of the turnover of formamidopyrimidine DNA glycosylase

In recent years, significant progress has been made in determining the catalytic mechanisms by which base excision repair (BER) DNA glycosylases and glycosylase-abasic site (AP) lyases cleave the glycosyl bond. While these investigations have identified active site residues and active site architectures, few investigations have analyzed postincision turnover events. Previously, we identified a critical residue (His16) in the T4-pyrimidine dimer glycosylase (T4-Pdg) that, when mutated, interferes with enzyme turnover [Meador et al. (2004) J. Biol. Chem. 279, 3348-3353]. To test whether comparable residues and mechanisms might be operative for other BER glycosylase:AP-lyases, molecular modeling studies were conducted comparing the active site regions of T4-Pdg and the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg). These analyses revealed that His71 in Fpg might perform a similar function to His16 in T4-Pdg. Site-directed mutagenesis of the Fpg gene and analyses of the reaction mechanism of the mutant enzyme revealed that the H71A enzyme retained activity on a DNA substrate containing an 8-oxo-7,8-dihydroguanine (8-oxoG) opposite cytosine and DNA containing an AP site. The H71A Fpg mutant was severely compromised in enzyme turnover on the 8-oxoG-C substrate but had turnover rates comparable to that of wild-type Fpg on AP-containing DNA. The similar mutant phenotypes for these two enzymes, despite a complete lack of structural or sequence homology between them, suggest a common mechanism for the rate-limiting step catalyzed by BER glycosylase:AP-lyases.     

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.     

hUNG2 is the major repair enzyme for removal of uracil from U:A matches,U:G mismatches,and U in single-stranded DNA,with hSMUG1 as a broad specificity backup

hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double- and single-stranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg2+, at which the activity of hUNG2 is 2-3 orders of magnitude higher than of hSMUG1. Moreover, Mg2+ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N(4)-ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup.     

Gelonin is an unusual DNA glycosylase that removes adenine from single-stranded DNA, normal base pairs and mismatches

We reported that plant ribosome inactivating proteins (RIP) have a unique DNA glycosylase activity that removes adenine from single-stranded DNA (Nicolas, E., Beggs, J. M., Haltiwanger, B. M., and Taraschi, T. F. (1998) J. Biol. Chem. 273, 17216-17220). In this investigation, we further characterized the interaction of the RIP gelonin with single-stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physiological pH, zinc and beta-mercaptoethanol stimulated the adenine DNA glycosylase activity of gelonin. Under these conditions, gelonin catalytically removed adenine from single-stranded DNA and, albeit to a lesser extent, from normal base pairs and mismatches in duplex DNA. Also unprecedented was the finding that activity on single-stranded and double-stranded oligonucleotides containing multiple adenines generated unstable products with several abasic sites, producing strand breakage and duplex melting, respectively. The results from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A re-examination of the classification of gelonin as a DNA glycosylase/AP lyase using the borohydride trapping assay revealed that gelonin was similar to the DNA glycosylase MutY: both enzymes are monofunctional glycosylases, which are trappable to their DNA substrates. The k(cat) for the removal of adenine from single-stranded DNA was close to the values observed with multisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA may be physiologically relevant.     

The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans

DNA from Kinetoplastida contains the unusual modified base beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fasciculata and Leishmania tarentolae. We purified the 90 kDa C.fasciculata J-binding protein 50 000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and L.tarentolae. Recombinant proteins expressed in Escherichia coli demonstrated J-specific DNA binding. The J-binding proteins show 43-63% identity and are unlike any known protein. The discovery of a J-binding protein suggests that J, like methylated cytosine in higher eukaryotes, functions via a protein intermediate.