Chemical approaches toward understanding base excision DNA repair

Despite the importance of DNA repair in protecting the genome, the molecular basis for damage recognition and repair remains poorly understood. In the base excision repair pathway (BER), DNA glycosylases recognize and excise damaged bases from DNA. This review focuses on the recent development of chemical approaches that have been applied to the study of BER enzymes. Several distinctive classes of noncleavable substrate analogs that form stable complexes with DNA glycosylases have recently been designed and synthesized. These analogs have been used for biochemical and structural analyses of protein—DNA complexes involving DNA glycosylases, and for the isolation of a novel DNA glycosylase. An approach to trap covalently a DNA glycosylase-intermediate complex has also been used to elucidate the mechanism of DNA glycosylases.     

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.     

Product-assisted catalysis in base-excision DNA repair

Most spontaneous damage to bases in DNA is corrected through the action of the base-excision DNA repair pathway. Base excision repair is initiated by DNA glycosylases, lesion-specific enzymes that intercept aberrant bases in DNA and catalyze their excision. How such proteins accomplish the feat of catalyzing no fewer than five sequential reaction steps using a single active site has been unknown. To help answer this, we report the structure of a trapped catalytic intermediate in DNA repair by human 8-oxoguanine DNA glycosylase. This structure and supporting biochemical results reveal that the enzyme sequesters the excised lesion base and exploits it as a cofactor to participate in catalysis. To our knowledge, the present example represents the first documented case of product-assisted catalysis in an enzyme-catalyzed reaction.     

Diverse functions of DNA glycosylases processing oxidative base lesions in brain

Endogenous and exogenous oxidative agents continuously damage genomic DNA, with the brain being particularly vulnerable. Thus, preserving genomic integrity is key for brain health and neuronal function. Accumulation of DNA damage is one of the causative factors of ageing and increases the risk of a wide range of neurological disorders. Base excision repair is the major pathway for removal of oxidized bases in the genome and initiated by DNA glycosylases. Emerging evidence suggest that DNA glycosylases have non-canonical functions important for genome regulation. Understanding canonical and non-canonical functions of DNA glycosylases processing oxidative base lesions modulating brain function will be crucial for the development of novel therapeutic strategies.     

DNA glycosylase recognition and catalysis

DNA glycosylases are the enzymes responsible for recognizing base lesions in the genome and initiating base excision DNA repair. Recent structural and biochemical results have provided novel insights into DNA damage recognition and repair. The basis of the recognition of the oxidative lesion 8-oxoguanine by two structurally unrelated DNA glycosylases is now understood and has been revealed to involve surprisingly similar strategies. Work on MutM (Fpg) has produced structures representing three discrete reaction steps. The NMR structure of 3-methyladenine glycosylase I revealed its place among the structural families of DNA glycosylases and the X-ray structure of SMUG1 likewise confirmed that this protein is a member of the uracil DNA glycosylase superfamily. A novel disulfide cross-linking strategy was used to obtain the long-anticipated structure of MutY bound to DNA containing an A*oxoG mispair.     

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.     

Base-excision repair of oxidative DNA damage by DNA glycosylases

Oxidative damage to DNA caused by free radicals and other oxidants generate base and sugar damage, strand breaks, clustered sites, tandem lesions and DNA-protein cross-links. Oxidative DNA damage is mainly repaired by base-excision repair in living cells with the involvement of DNA glycosylases in the first step and other enzymes in subsequent steps. DNA glycosylases remove modified bases from DNA, generating an apurinic/apyrimidinic (AP) site. Some of these enzymes that remove oxidatively modified DNA bases also possess AP-lyase activity to cleave DNA at AP sites. DNA glycosylases possess varying substrate specificities, and some of them exhibit cross-activity for removal of both pyrimidine- and purine-derived lesions. Most studies on substrate specificities and excision kinetics of DNA glycosylases were performed using oligonucleotides with a single modified base incorporated at a specific position. Other studies used high-molecular weight DNA containing multiple pyrimidine- and purine-derived lesions. In this case, substrate specificities and excision kinetics were found to be different from those observed with oligonucleotides. This paper reviews substrate specificities and excision kinetics of DNA glycosylases for removal of pyrimidine- and purine-derived lesions in high-molecular weight DNA.     

5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.     

Selective base excision repair of DNA damage by the non‐base‐flipping DNA glycosylase AlkC

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT‐like repeat (HLR) fold. AlkD uses a unique non‐base‐flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3‐methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non‐base‐flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin‐like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3‐methylcytosine (3mC) and N1‐methyladenine (1mA), which are also repaired by AlkB‐catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.     

Radiation-induced DNA damage and its repair

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes. A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment 32P-labelled at one end, it has been shown that the alkali-labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.     

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.     

3-methyladenine DNA glycosylases: structure,function, and biological importance

The genome continuously suffers damage due to its reactivity with chemical and physical agents. Finding such damage in genomes (that can be several million to several billion nucleotide base pairs in size) is a seemingly daunting task. 3-Methyladenine DNA glycosylases can initiate the base excision repair (BER) of an extraordinarily wide range of substrate bases. The advantage of such broad substrate recognition is that these enzymes provide resistance to a wide variety of DNA damaging agents; however, under certain circumstances, the eclectic nature of these enzymes can confer some biological disadvantages. Solving the X-ray crystal structures of two 3-methyladenine DNA glycosylases, and creating cells and animals altered for this activity, contributes to our understanding of their enzyme mechanism and how such enzymes influence the biological response of organisms to several different types of DNA damage. BioEssays 21:668–676, 1999. © 1999 John Wiley & Sons, Inc.     

Structural insight into repair of alkylated DNA by a new superfamily of DNA glycosylases comprising HEAT-like repeats

3-methyladenine DNA glycosylases initiate repair of cytotoxic and promutagenic alkylated bases in DNA. We demonstrate by comparative modelling that Bacillus cereus AlkD belongs to a new, fifth, structural superfamily of DNA glycosylases with an alpha-alpha superhelix fold comprising six HEAT-like repeats. The structure reveals a wide, positively charged groove, including a putative base recognition pocket. This groove appears to be suitable for the accommodation of double-stranded DNA with a flipped-out alkylated base. Site-specific mutagenesis within the recognition pocket identified several residues essential for enzyme activity. The results suggest that the aromatic side chain of a tryptophan residue recognizes electron-deficient alkylated bases through stacking interactions, while an interacting aspartate-arginine pair is essential for removal of the damaged base. A structural model of AlkD bound to DNA with a flipped-out purine moiety gives insight into the catalytic machinery for this new class of DNA glycosylases.     

Release of normal bases from intact DNA by a native DNA repair enzyme.

Base excision repair is initiated by DNA glycosylases removing inappropriate bases from DNA. One group of these enzymes, comprising 3-methyladenine DNA glycosylase II (AlkA) from Escherichia coli and related enzymes from other organisms, has been found to have an unusual broad specificity towards quite different base structures. We tested whether such enzymes might also be capable of removing normal base residues from DNA. The native enzymes from E.coli, Saccharomyces cerevisiae and human cells promoted release of intact guanines with significant frequencies, and further analysis of AlkA showed that all the normal bases can be removed. Transformation of E. coli with plasmids expressing different levels of AlkA produced an increased spontaneous mutation frequency correlated with the expression levels, indicating that excision of normal bases occurs at biologically significant rates. We propose that the broad specificity 3-methyladenine DNA glycosylases represent a general type of repair enzyme 'pulling' bases in DNA largely at random, without much preference for a specific structure. The specificity for release of damaged bases occurs because base structure alterations cause instability of the base-sugar bonds. Damaged bases are therefore released more readily than normal bases once the bond activation energy is reduced further by the enzyme. Qualitatively, the model correlates quite well with the relative rate of excision observed for most, if not all, of the substrates described for AlkA and analogues.     

Protective action of melatonin against oxidative DNA damage: chemical inactivation versus base-excision repair

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 microM and its direct metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 microM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.     

A role for iron-sulfur clusters in DNA repair

The presence of 4Fe-4S clusters in enzymes involved in DNA repair has posed the question of the role of these intricate cofactors in damaged DNA recognition and repair. It is particularly intriguing that base excision repair glycosylases that remove a wide variety of damaged bases, and also have vastly different sequences and structures, have been found to contain this cofactor. The accumulating biochemical and structural evidence indicates that the region supported by the cluster is intimately involved in DNA binding, and that such binding interactions impact catalysis of base removal. Recent evidence has also established that binding of the glycosylases to DNA facilitates oxidation of the [4Fe-4S](2+) cluster to the [4Fe-4S](3+) form. Notably, the measured redox potentials for a variety of 4Fe-4S cluster-containing glycosylases are remarkably similar. Based on this DNA-mediated redox behavior, it has been suggested that this property may be used to enhance the activity of these enzymes by facilitating damaged DNA location.     

Measurement of oxidatively generated base damage in cellular DNA

This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.     

Protective action of melatonin against oxidative DNA damage—Chemical inactivation versus base-excision repair

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 μM and its direct metabolite N¹-acetyl-N²-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 μM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.