A novel uracil-DNA glycosylase with broad substrate specificity and an unusual active site

Uracil-DNA glycosylases (UDGs) catalyse the removal of uracil by flipping it out of the double helix into their binding pockets, where the glycosidic bond is hydrolysed by a water molecule activated by a polar amino acid. Interestingly, the four known UDG families differ in their active site make-up. The activating residues in UNG and SMUG enzymes are aspartates, thermostable UDGs resemble UNG-type enzymes, but carry glutamate rather than aspartate residues in their active sites, and the less active MUG/TDG enzymes contain an active site asparagine. We now describe the first member of a fifth UDG family, Pa-UDGb from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum, the active site of which lacks the polar residue that was hitherto thought to be essential for catalysis. Moreover, Pa-UDGb is the first member of the UDG family that efficiently catalyses the removal of an aberrant purine, hypoxanthine, from DNA. We postulate that this enzyme has evolved to counteract the mutagenic threat of cytosine and adenine deamination, which becomes particularly acute in organisms living at elevated temperatures.     

Biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum

Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G.U and G.T mispairs, respectively, that must be restored to G.C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G.C --> A.T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G.U and G.T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G.U mispairs, in A.U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a approximately 22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinant Pa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus and Thermotoga maritima. Interestingly, neither Pa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.     

Base excision repair and its role in maintaining genome stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10(4) events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Repair of deaminated bases in DNA

Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil, hypoxanthine, and xanthine. When cells are under oxidative stress that is induced either by oxidizing agents or by mitochondrial dysfunction, additional deamination products such as 5-hydroxymethyluracil (5-HMU) and 5-hydroxyuracil (5-OH-Ura) are formed. The cellular level of these highly mutagenic lesions is increased substantially when cells are exposed to DNA damaging agent, such as ionizing radiation, redox reagents, nitric oxide, and others. The cellular repair of deamination products is predominantly through the base excision repair (BER) pathway, a major cellular repair pathway that is initiated by lesion specific DNA glycosylases. In BER, the lesions are removed by the combined action of a DNA glycosylase and an AP endonuclease, leaving behind a one-base gap. The gapped product is then further repaired by the sequential action of DNA polymerase and DNA ligase. DNA glycosylases that recognize uracil, 5-OH-Ura, 5-HMU (derived from 5-methylcytosine) and a T/G mismatch (derived from a 5-methylcytosine/G pair) are present in most cells. Many of these glycosylases have been cloned and well characterized. In yeast and mammalian cells, hypoxanthine is efficiently removed by methylpurine N-glycosylase, and it is thought that BER might be an important pathway for the repair of hypoxanthine. In contrast, no glycosylase that can recognize xanthine has been identified in either yeast or mammalian cells. In Escherichia coli, the major enzyme activity that initiates the repair of hypoxanthine and xanthine is endonuclease V. Endonuclease V is an endonuclease that hydrolyzes the second phosphodiester bond 3' to the lesion. It is hypothesized that the cleaved DNA is further repaired through an alternative excision repair (AER) pathway that requires the participation of either a 5' endonuclease or a 3'-5' exonuclease to remove the damaged base. The repair process is then completed by the sequential actions of DNA polymerase and DNA ligase. Endonuclease V sequence homologs are present in all kingdoms, and it is conceivable that endonuclease V might also be a major enzyme that initiates the repair of hypoxanthine and xanthine in mammalian cells.     

Base Excision Repair and its Role in Maintaining Genome Stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10⁴ events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Uracil-DNA glycosylase of Thermoplasma acidophilum directs long-patch base excision repair, which is promoted by deoxynucleoside triphosphates and ATP/ADP, into short-patch repair

Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilum and the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5'-acting AP endonuclease and a 5'-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3'-acting AP lyase, followed by a 3'-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilumcell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ～15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid single-nucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.     

Base excision repair

Damage to DNA bases resulting from deamination, oxidation, and alkylation is mainly repaired by base-excision repair. BER is initiated by DNA glycosylases, which recognize damaged bases and excise them from DNA by hydrolyzing the N-glycosidic bond between the base and the sugar phosphate backbone of DNA to generate an abasic site. Different human and E. coli DNA glycosylases have been cloned and characterized, each one with unique substrate specificity. Some of them additionally have AP lyase activity, which enables them to cleave the bond between the sugar and phosphate 3' to the damaged site. BER consist of two repair pathways (short or long) in which one or more nucleotides are introduced respectively. In conclusion, it seems to be likely that BER pathways are essential for genomic repair and stability in living cells.     

The rate of base excision repair of uracil is controlled by the initiating glycosylase

Uracil in DNA is repaired by base excision repair (BER) initiated by a DNA glycosylase, followed by strand incision, trimming of ends, gap filling and ligation. Uracil in DNA comes in two distinct forms; U:A pairs, typically resulting from replication errors, and mutagenic U:G mismatches, arising from cytosine deamination. To identify proteins critical to the rate of repair of these lesions, we quantified overall repair of U:A pairs, U:G mismatches and repair intermediates (abasic sites and nicked abasic sites) in vitro. For this purpose we used circular DNA substrates and nuclear extracts of eight human cell lines with wide variation in the content of BER proteins. We identified the initiating uracil-DNA glycosylase UNG2 as the major overall rate-limiting factor. UNG2 is apparently the sole glycosylase initiating BER of U:A pairs and generally initiated repair of almost 90% of the U:G mismatches. Surprisingly, TDG contributed at least as much as single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1) to BER of U:G mismatches. Furthermore, in a cell line that expressed unusually high amounts of TDG, this glycosylase contributed to initiation of as much as approximately 30% of U:G repair. Repair of U:G mismatches was generally faster than that of U:A pairs, which agrees with the known substrate preference of UNG-type glycosylases. Unexpectedly, repair of abasic sites opposite G was also generally faster than when opposite A, and this could not be explained by the properties of the purified APE1 protein. It may rather reflect differences in substrate recognition or repair by different complex(es). Lig III is apparently a minor rate-regulator for U:G repair. APE1, Pol beta, Pol delta, PCNA, XRCC1 and Lig I did not seem to be rate-limiting for overall repair of any of the substrates. These results identify damaged base removal as the major rate-limiting step in BER of uracil in human cells.     

Repair of oxidative DNA damage

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.     

Base excision repair of DNA in mammalian cells

Base excision repair (BER) of DNA corrects a number of spontaneous and environmentally induced genotoxic or miscoding base lesions in a process initiated by DNA glycosylases. An AP endonuclease cleaves at the 5' side of the abasic site and the repair process is subsequently completed via either short patch repair or long patch repair, which largely require different proteins. As one example, the UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) uracil DNA glycosylase and prevents accumulation of uracil in the genome. BER is likely to have a major role in preserving the integrity of DNA during evolution and may prevent cancer.     

Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover

DNA glycosylases initiate base excision repair (BER) through the generation of potentially harmful abasic sites (AP sites) in DNA. Human thymine-DNA glycosylase (TDG) is a mismatch-specific uracil/thymine-DNA glycosylase with an implicated function in the restoration of G*C base pairs at sites of cytosine or 5-methylcytosine deamination. The rate-limiting step in the action of TDG in vitro is its dissociation from the product AP site, suggesting the existence of a specific enzyme release mechanism in vivo. We show here that TDG interacts with and is covalently modified by the ubiquitin-like proteins SUMO-1 and SUMO-2/3. SUMO conjugation dramatically reduces the DNA substrate and AP site binding affinity of TDG, and this is associated with a significant increase in enzymatic turnover in reactions with a G*U substrate and the loss of G*T processing activity. Sumoylation also potentiates the stimulatory effect of APE1 on TDG. These observations implicate a function of sumoylation in the controlled dissociation of TDG from the AP site and open up novel perspectives for the understanding of the molecular mechanisms coordinating the early steps of BER.     

Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase

The XPC-HR23B complex recognizes various helix-distorting lesions in DNA and initiates global genome nucleotide excision repair. Here we describe a novel functional interaction between XPC-HR23B and thymine DNA glycosylase (TDG), which initiates base excision repair (BER) of G/T mismatches generated by spontaneous deamination of 5-methylcytosine. XPC-HR23B stimulated TDG activity by promoting the release of TDG from abasic sites that result from the excision of mismatched T bases. In the presence of AP endonuclease (APE), XPC-HR23B had an additive effect on the enzymatic turnover of TDG without significantly inhibiting the subsequent action of APE. Our observations suggest that XPC-HR23B may participate in BER of G/T mismatches, thereby contributing to the suppression of spontaneous mutations that may be one of the contributory factors for the promotion of carcinogenesis in xeroderma pigmentosum genetic complementation group C patients.     

Characterization of a thermostable DNA glycosylase specific for U/G and T/G mismatches from the hyperthermophilic archaeon Pyrobaculum aerophilum

U/G and T/G mismatches commonly occur due to spontaneous deamination of cytosine and 5-methylcytosine in double-stranded DNA. This mutagenic effect is particularly strong for extreme thermophiles, since the spontaneous deamination reaction is much enhanced at high temperature. Previously, a U/G and T/G mismatch-specific glycosylase (Mth-MIG) was found on a cryptic plasmid of the archaeon Methanobacterium thermoautotrophicum, a thermophile with an optimal growth temperature of 65 degrees C. We report characterization of a putative DNA glycosylase from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The open reading frame was first identified through a genome sequencing project in our laboratory. The predicted product of 230 amino acids shares significant sequence homology to [4Fe-4S]-containing Nth/MutY DNA glycosylases. The histidine-tagged recombinant protein was expressed in Escherichia coli and purified. It is thermostable and displays DNA glycosylase activities specific to U/G and T/G mismatches with an uncoupled AP lyase activity. It also processes U/7,8-dihydro-oxoguanine and T/7,8-dihydro-oxoguanine mismatches. We designate it Pa-MIG. Using sequence comparisons among complete bacterial and archaeal genomes, we have uncovered a putative MIG protein from another hyperthermophilic archaeon, Aeropyrum pernix. The unique conserved amino acid motifs of MIG proteins are proposed to distinguish MIG proteins from the closely related Nth/MutY DNA glycosylases.     

Lessons learned from structural results on uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG) functions as a sentry guarding against uracil in DNA. UDG initiates DNA base excision repair (BER) by hydrolyzing the uracil base from the deoxyribose. As one of the best studied DNA glycosylases, a coherent and complete functional mechanism is emerging that combines structural and biochemical results. This functional mechanism addresses the detection of uracil bases within a vast excess of normal DNA, the features of the enzyme that drive catalysis, and coordination of UDG with later steps of BER while preventing the release of toxic intermediates. Many of the solutions that UDG has evolved to overcome the challenges of policing the genome are shared by other DNA glycosylases and DNA repair enzymes, and thus appear to be general.     

Preferential CEBP binding to T:G mismatches and increased C-to-T human somatic mutations

DNA cytosine methylation in mammals modulates gene expression and chromatin accessibility. It also impacts mutation rates, via spontaneous oxidative deamination of 5-methylcytosine (5mC) to thymine. In most cases the resulting T:G mismatches are repaired, following T excision by one of the thymine DNA glycosylases, TDG or MBD4. We found that C-to-T mutations are enriched in the binding sites of CCAAT/enhancer binding proteins (CEBP). Within a CEBP site, the presence of a T:G mismatch increased CEBPβ binding affinity by a factor of >60 relative to the normal C:G base pair. This enhanced binding to a mismatch inhibits its repair by both TDG and MBD4 in vitro. Furthermore, repair of the deamination product of unmethylated cytosine, which yields a U:G DNA mismatch that is normally repaired via uracil DNA glycosylase, is also inhibited by CEBPβ binding. Passage of a replication fork over either a T:G or U:G mismatch, before repair can occur, results in a C-to-T mutation in one of the daughter duplexes. Our study thus provides a plausible mechanism for accumulation of C-to-T human somatic mutations.     

Base excision repair in yeast and mammals

Base excision repair (BER), as initiated by at least seven different DNA glycosylases or by enzymes that cleave DNA at abasic sites, executes the repair of a wide variety of DNA damages. Many of these damages arise spontaneously because DNA interacts with the cellular milieu, and so BER profoundly influences spontaneous mutation rates. In addition, BER provides significant protection against the toxic and mutagenic effects of DNA damaging agents present in the external environment, and as such is likely to prevent the adverse health effects of such agents. BER pathways have been studied in a wide variety of organisms (including yeasts) and here we review how these varied studies have shaped our current view of human BER.     

Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2

8-Oxoguanine (8-oxoG) is an unstable mutagenic DNA lesion that is prone to further oxidation. High valent metals such as Cr(V) and Ir(IV) readily oxidize 8-oxoG to form guanidinohydantoin (Gh), its isomer iminoallantoin (Ia), and spiroiminodihydantoin (Sp). When present in DNA, these lesions show enhanced base misincorporation over the parent 8-oxoG lesion leading to G --> T and G --> C transversion mutations and polymerase arrest. These findings suggested that further oxidized lesions of 8-oxoG are more mutagenic and toxic than 8-oxoG itself. Repair of oxidatively damaged bases, including Sp and Gh/Ia, are initiated by the base excision repair (BER) system that involves the DNA glycosylases Fpg, Nei, and Nth in E. coli. Mammalian homologs of two of these BER enzymes, OGG1 and NTH1, have little or no affinity for Gh/Ia and Sp. Herein we report that two recently identified mammalian glycosylases, NEIL1 and NEIL2, showed a high affinity for recognition and cleavage of DNA containing Gh/Ia and Sp lesions. NEIL1 and NEIL2 recognized both of these lesions in single-stranded DNA and catalyzed the removal of the lesions through a beta- and delta-elimination mechanism. NEIL1 and NEIL2 also recognized and excised the Gh/Ia lesion opposite all four natural bases in double-stranded DNA. NEIL1 was able to excise the Sp lesion opposite the four natural bases in double-stranded DNA, however, NEIL2 showed little cleavage activity against the Sp lesion in duplex DNA although DNA trapping studies show recognition and binding of NEIL2 to this lesion. This work suggests that NEIL1 and NEIL2 are essential in the recognition of further oxidized lesions arising from 8-oxoG and implies that these BER glycosylases may play an important role in the repair of DNA damage induced by carcinogenic metals.     

Stability,miscoding potential,and repair of 2'-deoxyxanthosine in DNA: implications for nitric oxide-induced mutagenesis

Nitric oxide (NO(*)) reacts with guanine in DNA and RNA to produce xanthine (X) as a major product. Despite its potential importance in NO(*)-mediated mutagenesis, the biochemical properties of X in polynucleotides have been relatively unexplored. We describe the synthesis and chemical characterization of xanthine-containing oligonucleotides and report on the susceptibility of X to depurination, its miscoding potential during replication by polymerases, and its recognition and excision by several members of the base excision repair (BER) family of DNA glycosylases. At neutral pH, X was found to be only slightly less stable than guanine to depurination (k(X)/k(G) = 1.19), whereas at pH Mpg > Nth > Fpg. Implications of these results for the induction of mutations by nitric oxide are discussed.     

Suppression of spontaneous mutagenesis in human cells by DNA base excision–repair

The chemical instability of the covalent structure of DNA, and in vivo exposure of DNA to reactive oxygen species and endogenously produced alkylating agents, has triggered the evolution of several specific DNA repair pathways. A major strategy of repair involves the initial removal of an altered base from DNA by a member of the enzyme family of DNA glycosylases. The currently known enzymes of this type in mammalian cells are reviewed, and the subsequent base excision–repair (BER) steps that achieve restoration of the intact DNA structure are also described. The specific problem of retaining high accuracy in this essentially error-free repair process is discussed.     

Substrate specificities and excision kinetics of DNA glycosylases involved in base-excision repair of oxidative DNA damage

Reactive oxygen-derived species such as free radicals are formed in living cells by normal metabolism and exogenous sources, and cause a variety of types of DNA damage such as base and sugar damage, strand breaks and DNA-protein cross-links. Living organisms possess repair systems that repair DNA damage. Oxidative DNA damage caused by free radicals and other oxidizing agents is mainly repaired by base-excision repair (BER), which involves DNA glycosylases in the first step of the repair process. These enzymes remove modified bases from DNA by hydrolyzing the glycosidic bond between the modified base and the sugar moiety, generating an apurinic/apyrimidinic (AP) site. Some also possess AP lyase activity that subsequently cleaves DNA at AP sites. Many DNA glycosylases have been discovered and isolated, and their reaction mechanisms and substrate specificities have been elucidated. Most of the known products of oxidative damage to DNA are substrates of DNA glycosylases with broad or narrow substrate specificities. Some possess cross-activity and remove both pyrimidine- and purine-derived lesions. Overlapping activities between enzymes also exist. Studies of substrate specificities have been performed using either oligodeoxynucleotides with a single modified base embedded at a specific position or damaged DNA substrates containing a multiplicity of pyrimidine- and purine-derived lesions. This paper reviews the substrate specificities and excision kinetics of DNA glycosylases that have been investigated with the use of gas chromatography/mass spectrometry and DNA substrates with multiple lesions.