Oxidative base damage to DNA: specificity of base excision repair enzymes

Base excision repair (BER) is likely to be the main mechanism involved in the enzymatic restoration of oxidative base lesions within the DNA of both prokaryotic and eukaryotic cells. Emphasis was placed in early studies on the determination of the ability of several bacterial DNA N-glycosylases, including Escherichia coli endonuclease III (endo III) and formamidopyrimidine DNA N-glycosylase (Fpg), to recognize and excise several oxidized pyrimidine and purine bases. More recently, the availability of related DNA repair enzymes from yeast and human has provided new insights into the enzymatic removal of several.OH-mediated modified DNA bases. However, it should be noted that most of the earlier studies have involved globally modified DNA as the substrates. This explains, at least partly, why there is a paucity of accurate kinetic data on the excision rate of most of the modified bases. Interestingly, several oxidized pyrimidine and purine nucleosides have been recently inserted into defined sequence oligonucleotides. The use of the latter substrates, together with overexpressed DNA N-glycosylases, allows detailed studies on the efficiency of the enzymatic release of the modified bases. This was facilitated by the development of accurate chromatographic and mass spectrometric methods aimed at measuring oxidized bases and nucleosides. As one of the main conclusions, it appears that the specificity of both endo III and Fpg proteins is much broader than expected a few years ago.     

Cross-linking of 2-deoxyribonolactone and its beta-elimination product by base excision repair enzymes

2-Deoxyribonolactone (3) is produced in DNA as a result of reaction with a variety of DNA damaging agents. The lesion undergoes beta-elimination to form a second metastable electrophilic product (4). In this study, DNA containing 2-deoxyribonolactone (3) and its beta-elimination product (4) are generated at specific sites using a photolabile nucleotide precursor. 2-Deoxyribonolactone is not incised by any of the 8 AP lyases tested. One enzyme, Escherichia coli endonuclease III, cross-links to 3, and the lesion strongly inhibits excision of typical abasic sites by this enzyme. Two of the enzymes, FPG and NEIL1 known to cleave normal abasic sites (1) by effecting beta,delta-elimination form cross-links to the butenolide lesion (4). The observed results are ascribable to characteristics of the enzymes and the lesions. These enzymes are also important for the removal of oxidative base lesions. These results suggest that high concentrations of 3 and 4 may exert significant effects on the repair of normal AP site and oxidative base lesions in cells by reducing the cellular activity of these BER enzymes either via cross-linking or competing with binding to the BER enzymes.     

Action of multiple base excision repair enzymes on the 2'-deoxyribonolactone

Free radical attack on the sugar-phosphate backbone generates oxidized apurinic/apyrimidinic (AP) residues in DNA. 2'-deoxyribonolactone (dL) is a C1'-oxidized AP site damage generated by UV and gamma-irradiation, and certain anticancer drugs. If not repaired dL produces G-->A transitions in Escherichia coli. In the base excision repair (BER) pathway, AP endonucleases are the major enzymes responsible for 5'-incision of the regular AP site (dR) and dL. DNA glycosylases with associated AP lyase activity can also efficiently cleave regular AP sites. Here, we report that dL is a substrate for AP endonucleases but not for DNA glycosylases/AP lyases. The kinetic parameters of the dL-incision were similar to those of the dR. DNA glycosylases such as E. coli formamidopyrimidine-DNA glycosylase, mismatch-specific uracil-DNA glycosylase, and human alkylpurine-DNA N-glycosylase bind strongly to dL without cleaving it. We show that dL cross-links with the human proteins 8-oxoguanine-DNA (hOGG1) and thymine glycol-DNA glycosylases (hNth1), and dR cross-links with Nth and hNth1. These results suggest that dL and dR induced genotoxicity might be strengthened by BER pathway in vivo.     

Heat-shock proteins associated with base excision repair enzymes in HeLa cells

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.     

Regulation of base excision repair proteins by ubiquitylation

Human cellular DNA is under constant attack from both endogenous and exogenous mutagens, and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying complexity. BER thus maintains genome stability, and prevents the development of human diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.     

Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency

Single-base lesions in DNA are repaired predominantly by base excision repair (BER). DNA polymerase beta (pol beta) is the polymerase of choice in the preferred single-nucleotide BER pathway. The characteristic phenotype of mouse fibroblasts with a deletion of the pol beta gene is moderate hypersensitivity to monofunctional alkylating agents, e.g., methyl methanesulfonate (MMS). Increased sensitivity to MMS is also seen in the absence of pol beta partner proteins XRCC1 and PARP-1, and under conditions where BER efficiency is reduced by synthetic inhibitors. PARP activity plays a major role in protection against MMS-induced cytotoxicity, and cells treated with a combination of non-toxic concentrations of MMS and a PARP inhibitor undergo cell cycle arrest and die by a Chk1-dependent apoptotic pathway. Since BER-deficient cells and tumors are similarly hypersensitive to the clinically used chemotherapeutic methylating agent temozolomide, modulation of DNA damage-induced cell signaling pathways, as well as BER, are attractive targets for potentiating chemotherapy.     

Imbalanced base excision repair in response to folate deficiency is accelerated by polymerase beta haploinsufficiency

The mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase beta (beta-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce beta-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, beta-pol-haploinsufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young beta-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and beta-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for beta-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases.     

Repair activity of base and nucleotide excision repair enzymes for guanine lesions induced by nitrosative stress

Nitric oxide (NO) induces deamination of guanine, yielding xanthine and oxanine (Oxa). Furthermore, Oxa reacts with polyamines and DNA binding proteins to form cross-link adducts. Thus, it is of interest how these lesions are processed by DNA repair enzymes in view of the genotoxic mechanism of NO. In the present study, we have examined the repair capacity for Oxa and Oxa–spermine cross-link adducts (Oxa–Sp) of enzymes involved in base excision repair (BER) and nucleotide excision repair (NER) to delineate the repair mechanism of nitrosative damage to guanine. Oligonucleotide substrates containing Oxa and Oxa–Sp were incubated with purified BER and NER enzymes or cell-free extracts (CFEs), and the damage-excising or DNA-incising activity was compared with that for control (physiological) substrates. The Oxa-excising activities of Escherichia coli and human DNA glycosylases and HeLa CFEs were 0.2–9% relative to control substrates, implying poor processing of Oxa by BER. In contrast, DNA containing Oxa–Sp was incised efficiently by UvrABC nuclease and SOS-induced E.coli CFEs, suggesting a role of NER in ameliorating genotoxic effects associated with nitrosative stress. Analyses of the activity of CFEs from NER-proficient and NER-deficient human cells on Oxa–Sp DNA confirmed further the involvement of NER in the repair of nitrosative DNA damage.     

Arsenicals affect base excision repair by several mechanisms

Inorganic arsenic is a strong, widespread human carcinogen. How exactly inorganic arsenic exerts carcinogenicity in humans is as yet unclear, but it is thought to be closely related to its metabolism. At exposure-relevant concentrations arsenic is neither directly DNA reactive nor mutagenic. Thus, more likely epigenetic and indirect genotoxic effects, among others a modulation of the cellular DNA damage response and DNA repair, are important molecular mechanisms contributing to its carcinogenicity. In the present study, we investigated the impact of arsenic on several base excision repair (BER) key players in cultured human lung cells. For the first time gene expression, protein level and in case of human 8-oxoguanine DNA glycosylase 1 (hOGG1) protein function was examined in one study, comparing inorganic arsenite and its trivalent and pentavalent mono- and dimethylated metabolites, also taking into account their cellular bioavailability. Our data clearly show that arsenite and its metabolites can affect several cellular endpoints related to DNA repair. Thus, cellular OGG activity was most sensitively affected by dimethylarsinic acid (DMA(V)), DNA ligase IIIα (LIGIIIα) protein level by arsenite and X-ray cross complementing protein 1 (XRCC1 protein) content by monomethylarsonic acid (MMA(V)), with significant effects starting at ≥3.2μM cellular arsenic. With respect to MMA(V), to our knowledge these effects are the most sensitive endpoints, related to DNA damage response, that have been identified so far. In contrast to earlier nucleotide excision repair related studies, the trivalent methylated metabolites exerted strong effects on the investigated BER key players only at cytotoxic concentrations. In summary, our data point out that after mixed arsenic species exposure, a realistic scenario after oral inorganic arsenic intake in humans, DNA repair might be affected by different mechanisms and therefore very effectively, which might facilitate the carcinogenic process of inorganic arsenic.     

Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors

Lack of reliable assays for DNA repair has largely prevented measurements of DNA repair from being included in human biomonitoring studies. Using newly developed modifications of the comet assay we tested whether a fruit‐ and antioxidant‐rich plant‐based intervention could affect base excision repair (BER) and nucleotide excision repair (NER) in a group of 102 male volunteers. BER and NER repair capacities were measured in lymphocytes before and after a dietary intervention lasting 8 weeks. The study had one control group, one group consuming three kiwifruits per day and one group consuming a variety of antioxidant‐rich fruits and plant products in addition to their normal diet. DNA strand breaks were reduced following consumption of both kiwifruits (13%, p = 0.05) and antioxidant‐rich plant products (20%, p = 0.02). Increased BER (55%, p = 0.01) and reduced NER (?39%, p < 0.01) were observed in the group consuming a wide variety of plant products. Reduced NER was also observed in the kiwifruit group (?38%, p = 0.05), but BER was not affected in this group. Here we have demonstrated that DNA repair is affected by diet and that modified versions of the comet assay can be used to assess activity of different DNA repair pathways in human biomonitoring studies. Copyright © 2010 John Wiley & Sons, Ltd.     

Variation in base excision repair capacity

The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence variability in DNA repair genes and several studies have associated BER gene polymorphisms with disease risk, including cancer of several sites. The intent of this review is to describe the range of BER capacity among individuals and the functional consequences of BER genetic variants. We also discuss studies that associate BER deficiency with disease risk and the current state of BER capacity measurement assays.     

CHIPping away at base excision repair

In this issue of Molecular Cell, Parsons et al. (2008) report that the E3 ubiquitin ligase CHIP regulates the stability of the base excision repair (BER) proteins XRCC1 and DNA Pol beta, adding a new level of regulation for BER.     

Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. MPG activity and other DNA glycosylases do not have an absolute requirement for a cofactor. In contrast, all downstream activities of major base excision repair proteins, such as apurinic/apyrimidinic endonuclease, DNA polymerase beta, and ligases, require Mg(2+). Here we have demonstrated that Mg(2+) can be significantly inhibitory toward MPG activity depending on its concentration but independent of substrate type. The pre-steady-state kinetics suggests that Mg(2+) at high but physiologic concentrations decreases the amount of active enzyme concentrations. Steady-state inhibition kinetics showed that Mg(2+) affected K(m), but not V(max), and the inhibition could be reversed by EDTA but not by DNA. At low concentration, Mg(2+) stimulated the enzyme activity only with hypoxanthine but not ethenoadenine. Real-time binding experiments using surface plasmon resonance spectroscopy showed that the pronounced inhibition of activity was due to inhibition in substrate binding. Nonetheless, the glycosidic bond cleavage step was not affected. These results altogether suggest that Mg(2+) inhibits MPG activity by abrogating substrate binding. Because Mg(2+) is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.     

Mechanistic comparisons among base excision repair glycosylases

The mechanisms by which various DNA glycosylases initiate the base excision repair pathways are discussed. Fundamental distinctions are made between "simple glycosylases," that do not form DNA single-strand breaks, and "glycosylases/abasic site lyases," that do form single-strand breaks. Several groupings of BER substrate sites are defined and some interactions between these groupings and glycosylase mechanisms discussed. Two characteristics are proposed to be common among all BER glycosylases: a nucleotide flipping step that serves to expose the scissile glycosyl bond to catalysis, and a glycosylase transition state characterized by substantial tetrahedral character at the base glycosyl atom.     

BRCA1 regulation of base excision repair pathway

This feature item briefly reviews the relation of the tumor suppressor function of BRCA1 with DNA damage and repair. The main focus of this article is on the regulation of base excision repair (BER) pathway by BRCA1 and in that context we introduce poly (ADP-ribose) polymerase 1 (PARP-1), which is a key enzyme in BER pathway, and its possible regulation by BRCA1.     

Co-ordination of base excision repair and genome stability

Base excision repair (BER) is a major DNA repair pathway employed in mammalian cells that is required to maintain genome stability, thus preventing several human diseases, such as ageing, neurodegenerative diseases and cancer. This is achieved through the repair of damaged DNA bases, sites of base loss and single strand breaks of varying complexity that are continuously induced endogenously or via exogenous mutagens. Whilst the enzymes involved in BER are now well known and characterised, the role of the co-ordination of BER enzymatic activities in the cellular response to DNA damage and the mechanisms regulating this process are only now being revealed. Post-translational modifications of BER proteins, including ubiquitylation and phosphorylation, are increasingly being identified as key processes that regulate BER. In this review we will summarise recent evidence discovering novel mechanisms that are involved in maintaining genome stability by regulation of the key BER proteins in response to DNA damage.