Action Of DNA Glycosylases Research Articles

The Kinetic Mechanism of 3'-5' Exonucleolytic Activity of AP Endonuclease Nfo from E. coli.

Escherichia coli apurinic/apyrimidinic (AP) endonuclease Nfo is one of the key participants in DNA repair. The principal biological role of this enzyme is the recognition and hydrolysis of AP sites, which arise in DNA either as a result of the spontaneous hydrolysis of an N-glycosidic bond with intact nitrogenous bases or under the action of DNA glycosylases, which eliminate various damaged bases during base excision repair. Nfo also removes 3′-terminal blocking groups resulting from AP lyase activity of DNA glycosylases. Additionally, Nfo can hydrolyze the phosphodiester linkage on the 5′ side of some damaged nucleotides on the nucleotide incision repair pathway. The function of 3′-5′-exonuclease activity of Nfo remains unclear and probably consists of participation (together with the nucleotide incision repair activity) in the repair of cluster lesions. In this work, using polyacrylamide gel electrophoresis and the stopped-flow method, we analyzed the kinetics of the interaction of Nfo with various model DNA substrates containing a 5′ single-stranded region. These data helped to describe the mechanism of nucleotide cleavage and to determine the rates of the corresponding stages. It was revealed that the rate-limiting stage of the enzymatic process is a dissociation of the reaction product from the enzyme active site. The stability of the terminal pair of nucleotides in the substrate did not affect the enzymatic-reaction rate. Finally, it was found that 2′-deoxynucleoside monophosphates can effectively inhibit the 3′-5′-exonuclease activity of Nfo.

Design and activity of AP endonuclease-1 inhibitors.

Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1) is a critical component of base excision repair that excises abasic lesions created enzymatically by the action of DNA glycosylases on modified bases and non-enzymatically by hydrolytic depurination/depyrimidination of nucleobases. Many anticancer drugs generate DNA adducts that are processed by base excision repair, and tumor resistance is frequently associated with enhanced APE-1 expression. Accordingly, APE-1 is a potential therapeutic target to treat cancer. Using computational approaches and the high resolution structure of APE-1, we developed a 5-point pharmacophore model for APE-1 small molecule inhibitors. One of the nM APE-1 inhibitors (AJAY-4) that was identified based on this model exhibited an overall median growth inhibition (GI50) of 4.19μM in the NCI-60 cell line panel. The mechanism of action is shown to be related to the buildup of abasic sites that cause PARP activation and PARP cleavage, and the activation of caspase-3 and caspase-7, which is consistent with cell death by apoptosis. In a drug combination growth inhibition screen conducted in 10 randomly selected NCI-60 cell lines and with 20 clinically used non-genotoxic anticancer drugs, a synergy was flagged in the SK-MEL-5 melanoma cell line exposed to combinations of vemurafenib, which targets melanoma cells with V600E mutated BRAF, and AJAY-4, our most potent APE-1 inhibitor. The synergy between AJAY-4 and vemurafenib was not observed in cell lines expressing wild-type B-Raf protein. This synergistic combination may provide a solution to the resistance that develops in tumors treated with B-Raf-targeting drugs.

Global Genome Nucleotide Excision Repair Proteins Rhp7p and Rhp41p Are Involved in Abasic Site Repair of <i>Schizosaccharomyces pombe</i>

The roles of nucleotide excision repair (NER) proteins in removing UV-induced lesions are well defined. There are two distinct NER pathways: global genome NER (GG-NER) and transcriptioncoupled NER. In human GG-NER, two heteromeric protein complexes, DDB1-DDB2 and XPC-RAD23, are responsible for initial lesion recognition. Here, we examined the genetic interactions between GG-NER and base excision repair (BER) genes during abasic (AP) site repair of ＂Schizosaccharomyces pombe＂. Mutants of ＂rhp＂7 (＂rhp＂7-＂rhp＂16 are functional homologs of DDB1-DDB2) and ＂rhp＂41 (XPC homolog) were moderately sensitive to methyl methanesulfonate and slightly to sodium bisulfite. Nth1p most actively cleaves the AP site in ＂S. pombe＂. Deletion of ＂rhp＂7 or ＂rhp＂41 from ＂nth＂1Δ cells greatly increased their sensitivity to alkylation and deamination, indicating that Rhp7p and Rhp41p are involved in repair of the AP sites generated by the action of DNA glycosylase. Induction of ＂rhp＂7 and ＂rhp＂16 genes by different types of DNA damage supports the ability of GG-NER to remove non-bulky lesions. Therefore, GG-NER activity not only targets bulky DNA helix-distorting lesions, but can also efficiently remove AP sites synergistically with BER.

Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

In host cells, Mycobacterium tuberculosis encounters an array of reactive molecules capable of damaging its genome. Non-bulky DNA lesions are the most common damages produced on the exposure of the pathogen to reactive species and base excision repair (BER) pathway is involved in the repair of such damage. During BER, apurinic/apyrimidinic (AP) endonuclease enzymes repair the abasic sites that are generated after spontaneous DNA base loss or by the action of DNA glycosylases, which if left unrepaired lead to inhibition of replication and transcription. However, the role of AP endonucleases in imparting protection against DNA damage and in the growth and pathogenesis of M.tuberculosis has not yet been elucidated. To demonstrate the biological significance of these enzymes in M.tuberculosis, it would be desirable to disrupt the relevant genes and evaluate the resulting mutants for their ability to grow in the host and cause disease. In this study, we have generated M.tuberculosis mutants of the base excision repair (BER) system, disrupted in either one (MtbΔend or MtbΔxthA) or both the AP endonucleases (MtbΔendΔxthA). We demonstrate that these genes are crucial for bacteria to withstand alkylation and oxidative stress in vitro. In addition, the mutant disrupted in both the AP endonucleases (MtbΔendΔxthA) exhibited a significant reduction in its ability to survive inside human macrophages. However, infection of guinea pigs with either MtbΔend or MtbΔxthA or MtbΔendΔxthA resulted in the similar bacillary load and pathological damage in the organs as observed in the case of infection with wild-type M.tuberculosis. The implications of these observations are discussed.

Identification and Characterization of Human Apurinic/Apyrimidinic Endonuclease-1 Inhibitors

The repair of abasic sites that arise in DNA from hydrolytic depurination/depyrimidination of the nitrogenous bases from the sugar-phosphate backbone and the action of DNA glycosylases on deaminated, oxidized, and alkylated bases are critical to cell survival. Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1; aka APE1/ref-1) is responsible for the initial removal of abasic lesions as part of the base excision repair pathway. Deletion of APE-1 activity is embryonic lethal in animals and is lethal in cells. Potential inhibitors of the repair function of APE-1 were identified based upon molecular modeling of the crystal structure of the APE-1 protein. We describe the characterization of several unique nanomolar inhibitors using two complementary biochemical screens. The most active molecules all contain a 2-methyl-4-amino-6,7-dioxolo-quinoline structure that is predicted from the modeling to anchor the compounds in the endonuclease site of the protein. The mechanism of action of the selected compounds was probed by fluorescence and competition studies, which indicate, in a specific case, direct interaction between the inhibitor and the active site of the protein. It is demonstrated that the inhibitors induce time-dependent increases in the accumulation of abasic sites in cells at levels that correlate with their potency to inhibit APE-1 endonuclease excision. The inhibitor molecules also potentiate by 5-fold the toxicity of a DNA methylating agent that creates abasic sites. The molecules represent a new class of APE-1 inhibitors that can be used to probe the biology of this critical enzyme and to sensitize resistant tumor cells to the cytotoxicity of clinically used DNA damaging anticancer drugs.

Modulation of the activity of methyl binding domain protein 4 (MBD4/MED1) while processing iododeoxyuridine generated DNA mispairs

DNA glycosylases function to remove endogenous and exogenous base damageand thus contribute to the maintenance of genomic integrity. This function gains clinicalrelevance when base mispairs introduced by chemotherapy or radiosensitizing drugsbecome their substrate. This report describes the action of DNA glycosylases on themispairs generated by iododeoxyuridine (IUdR) – a radiosensitizer. A non-radioactivefluorescent dye-based in vitro glycosylase assay was employed to quantitatively measurethe enzymatic activities of functionally related DNA glycosylases on IUdR generatedmispairs including G:IU and A:IU. Thymine DNA glycosylase (TDG) and methylbinding domain protein 4 (MBD4/MED1) are found to act on G:IU (but not A:IU)mispairs and are functionally complementary to each other. However, uracil DNAglycosylase (UDG) does not show any activity on these mispairs. The methyl bindingdomain of MBD4/MED1 was found to specifically inhibit the activity of MBD4/MED1as well as the glycosylase domain, when the G:IU mispairs were located in a methylatedCpG context. However, inhibition of TDG activity on methylated G:IU mispairs by themethyl binding domain was not observed.

AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis

Oxidative stress is a principal cause of DNA damage, and mechanisms to repair this damage are among the most highly conserved of biological processes. Oxidative stress is also used by phagocytes to attack bacterial pathogens in defence of the host. We have identified and characterised two apurinic/apyrimidinic (AP) endonuclease paralogues in the human pathogen Neisseria meningitidis. The presence of multiple versions of DNA repair enzymes in a single organism is usually thought to reflect redundancy in activities that are essential for cellular viability. We demonstrate here that these two AP endonuclease paralogues have distinct activities in DNA repair: one is a typical Neisserial AP endonuclease (NApe), whereas the other is a specialised 3'-phosphodiesterase Neisserial exonuclease (NExo). The lack of AP endonuclease activity of NExo is shown to be attributable to the presence of a histidine side chain, blocking the abasic ribose-binding site. Both enzymes are necessary for survival of N. meningitidis under oxidative stress and during bloodstream infection. The novel functional pairing of NExo and NApe is widespread among bacteria and appears to have evolved independently on several occasions.

Nitric oxide-induced homologous recombination in Escherichia coli is promoted by DNA glycosylases.

Nitric oxide (NO*) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO* leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO* toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO* toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO*-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO*-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO*-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO*-induced homologous recombination.

Structure of an 11-mer DNA Duplex Containing the Carbocyclic Nucleotide Analog: 2′-Deoxyaristeromycin

2′-deoxyaristeromycin (dAr) is a nucleoside analogue that is resistant to the action of DNA glycosylases. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of an 11-mer DNA containing a single dAr•T base pair at its center. Analysis of the spectra revealed the existence of a right-handed duplex in solution, stabilized by Watson-Crick hydrogen bonding and base-stacking interactions. The carbocyclic sugar adopted a C1′-exo conformation and sugars of the 3′-flanking base pair had puckers in the O4′—endo range. The dAr•T base pair was mildly propeller twisted, and the dAr analogue showed a positive roll with the 3′-flanking base. Our findings indicate that the observed resistance of dAr-containing oligodeoxynucleotides to the catalytic action of DNA glycosylases relates to its electronic properties rather than structure, and validate the use of dAr and related carbocyclic nucleoside analogues for biological and structure/function relationship studies.

A new member of the endonuclease III family of DNA repair enzymes that removes methylated purines from DNA

DNA is constantly exposed to endogenous andexogenous alkylating agents that can modify its bases,resulting in mutagenesis in the absence of DNA repair [1,2]. Alkylation damage is removed by the action of DNA glycosylases, which initiate the base excision repair pathway and protect the sequence information of the genome [3–5]. We have identified a new class of methylpurine DNA glycosylase, designated MpgII, that is a member of the endonuclease III family of DNA repair enzymes. We expressed and purified MpgII from Thermotoga maritima and found that the enzyme releases both 7-methylguanine and 3-methyladenine from DNA.We cloned the MpgII genes from T. maritima and from Aquifex aeolicus and found that both genes could restore methylmethanesulfonate (MMS) resistance to Escherichia coli alkA tagA double mutants, which are deficient in the repair of alkylated bases. Analogous genes are found in other Bacteria and Archaea and appear to be the only genes coding for methylpurine DNA glycosylase activity in these organisms. MpgII is the fifth member of the endonuclease III family of DNA repair enzymes, suggesting that the endonuclease III protein scaffold has been modified during evolution to recognize and repair a variety of DNA damage.

What structural features determine repair enzyme specificity and mechanism in chemically modified DNA?

A crucial question in repair is how do enzymes recognize substrates. In surveying the relevant literature, it becomes evident that there are no rules which can be clearly applied. At this time it appears that uracil glycosylase is the only repair enzyme for which all the known substrates can be rationalized on the basis of chemical structure. When surveying the multiplicity of substrates for m3A-DNA glycosylase, it is difficult, on the basis of present knowledge, to explain why 1,N6-etheno-A (epsilon A) is as good a substrate, if not better, than m3A for which the enzyme is named. There is no apparent unifying chemical structure which is required for recognition. It should also be noted that many studies of the mechanism of m3A-DNA glycosylase only utilized-N-3- and N-7-alkylpurines. On this basis, an electron-deficient purine, and later pyrimidine, was considered to be the recognition signal. Since epsilon A and Hx do not fall in this class, this is one illustration of why exploring new substrates becomes important in elucidating enzyme mechanisms. Ubiquitous enzymes, such as 5'-AP endonucleases, are present in both prokaryotes and eukaryotes. The primary function is the same, i.e., repair of an AP site which occurs through natural processes or from the action of DNA glycosylases. There are, however, completely unrelated substrates such as the exocyclic adducts pBQ-dC and pBQ-dG. pBQ-dC is repaired by both the human HAP1 and E. coli Exo III and Endo IV, while pBQ-dG is only repaired by the E. coli enzymes. Yet, when repair of these adducts occurs, it is by the same unusual pathway which differs from the usual base excision repair mechanism. This finding may ultimately not be as unusual as it now seems. The understanding of substrate recognition by repair enzymes, which can have different repair pathways, is complex. For example, three exocyclic derivatives which each have either the same modification (1,N4-epsilon dA and 3,N4-epsilon dC) or the same base with different modifying groups (3,N4-epsilon dC and 3,N4-pBQ-dC) are repaired by three separate enzymes and two mechanism (Figure 9). Investigators have also reported that two separate enzymes and pathways can be found for simple adducts such as m6G and O4T. It is not clear why, for these adducts, both MGMT and excision repair can be utilized. This could be visualized as a "backup" system and may be more common than now known. We cannot think like an enzyme or vice versa. In the absence of enough necessary information, we can only be descriptive. What information is necessary for further understanding? (1) More detailed structural studies of adducts in defined oligonucleotides would be useful. (2) New substrates should be explored. For example, is the mechanism for PBQ-dC (and pBQ-dG) repair unique? This involves guesswork and intuition. (3) For the adducts mentioned in this Perspective and others, understanding enzyme/substrate recognition will be facilitated by cocrystallography and site-directed mutagenesis. (4) Genetic approaches, such as knockouts or targeted mutations in repair genes, should be expanded in order to focus on the physiological role of a specific enzyme. Above all: structure, structure, structure! Enzymologists, organic chemists, physical chemiste, X-ray crystallographers, and others must combine forces if the fundamental problems addressed here are to be understood.

Regulation of eukaryotic abasic endonucleases and their role in genetic stability.

Abasic (AP) sites in DNA arise from spontaneous reactions or the action of DNA glycosylases and represent a loss of genetic information. The AP sites can be mutagenic or cytotoxic, and their repair is initiated by class II AP endonucleases, which incise immediately 5' to AP sites. The main enzyme of S. cerevisiae. Apn1, provides cellular resistance to oxidants (e.g., H2O2) or alkylating agents, and limits the spontaneous mutation rate. AP endonucleases from other species can replace Apn1 function in yeast to different extents. We studied the main human enzyme, Ape, with respect to its incision specificity in vitro and the expression of the APE gene in vivo. The results suggest that Ape evolved to act preferentially on AP sites compared to deoxyribose fragments located at oxidative strand breaks and that the incision modes of Ape and Apn1 may be fundamentally different. We also defined the functional APE promoter, and showed that APE expression is transiently downregulated during the regeneration of epidermis after wounding. This latter effect may lead to a window of vulnerability for DNA damage and perhaps mutagenesis during the healing of epidermal and other wounds. Such unexpected effects on the expression of DNA repair enzymes need to be taken into account in analyzing the susceptibility of different tissues to carcinogens.

Enzymes involved in the repair of damaged DNA

The multitude of enzymes responsible for removing damaged nucleotides from DNA in an error-free manner is reviewed. The most direct mechanisms include enzymatically catalyzed photoreversal of cyclobutane dimers and the removal of the O 6-methylguanine adduct from alkylated DNA by an enzyme whose presence is dependent on adaptation. The direct removal of either damaged purines or pyrimidines or partial removal of photochemically induced diadducts is catalyzed by DNA glycosylases in the absence of phosphodiester bond hydrolysis. Incision of DNA containing apurinic or apyrimidinic sites arising either spontaneously or by the action of DNA glycosylases is catalyzed by specific endonucleases. The incision of DNA containing bulky adducts is attributed to a multigenically controlled uvr system in Escherichia coli. The mechanisms of damaged nucleotide excision and reinsertion of nucleotides are controlled by unique exonuclease functions in either direct or indirect association with DNA polymerases.