At Apurinic/apyrimidinic Sites Research Articles

Mass Spectrometry Evidence for Forming Schiff Base 3'-DNA-Histone Cross-Links from Abasic Sites in Vitro and in Human Cells.

Histones catalyze DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by the formation of reversible but long-lived DNA-protein cross-links at 3'-termini (3'-histone-DPCs). However, the chemical structures of 3'-histone-DPCs are not well characterized, and whether they are formed in cells is uncertain. In this study, we developed a liquid chromatography with tandem mass spectrometry workflow to characterize DPCs produced from the reaction of histones with AP sites and wish to report evidence that histones cross-link to incised AP sites via Schiff bases. We also demonstrated for the first time that 3'-histone-DPCs are produced endogenously in human embryonic kidney 293T cells.

Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1).

Alkbh1 is one of nine mammalian homologues of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5'-product. The studies described here extend the biochemical characterization of two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to those of other AP lyases. The α,β-unsaturated aldehyde of the 5'-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examined with a newly developed assay using a methylation-sensitive restriction endonuclease, and the enzymatic rate was found to be very low. Indeed, the demethylase activity was less than half that of the AP lyase activity when ALKBH1 samples were assayed using identical buffer conditions. The two enzymatic activities were examined using a series of site-directed variant proteins, revealing the presence of distinct but partially overlapping active sites for the two reactions. We postulate that the very low 6-methyl adenine oxygenase activity associated with ALKBH1 is unlikely to represent the major function of the enzyme in the cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.

APE1 Incision Activity at Abasic Sites in Tandem Repeat Sequences

Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains.

An additional C-terminal loop in endonuclease IV, an apurinic/apyrimidinic endonuclease, controls binding affinity to DNA

Endonuclease IV (EndoIV) is an endonuclease that acts at apurinic/apyrimidinic (AP) sites and is classified as either long-type or short-type. The crystal structures of representative types of EndoIV from Geobacillus kaustophilus and Thermus thermophilus HB8 were determined using X-ray crystallography. G. kaustophilus EndoIV (the long type) had a higher affinity for double-stranded DNA containing an AP-site analogue than T. thermophilus EndoIV (the short type). Structural analysis of the two different EndoIVs suggested that a C-terminal DNA-recognition loop that is only present in the long type contributes to its high affinity for AP sites. A mutation analysis showed that Lys267 in the C-terminal DNA-recognition loop plays an important role in DNA binding.

Small molecule inhibitors of DNA repair nuclease activities of APE1.

APE1 is a multifunctional protein that possesses several nuclease activities, including the ability to incise at apurinic/apyrimidinic (AP) sites in DNA or RNA, to excise 3'-blocking termini from DNA ends, and to cleave at certain oxidized base lesions in DNA. Pre-clinical and clinical data indicate a role for APE1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs, particularly monofunctional alkylators and antimetabolites. In an effort to improve the efficacy of therapeutic compounds, such as temozolomide, groups have begun to develop high-throughput screening assays and to identify small molecule inhibitors against APE1 repair nuclease activities. It is envisioned that such inhibitors will be used in combinatorial treatment paradigms to enhance the efficacy of DNA-interactive drugs that introduce relevant cytotoxic DNA lesions. In this review, we summarize the current state of the efforts to design potent and selective inhibitors against APE1 AP site incision activity.

Structure of the endonuclease IV homologue from Thermotoga maritima in the presence of active-site divalent metal ions.

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5'-phosphodiester bond at an AP site to generate a free 3'-hydroxyl group and a 5'-terminal sugar phosphate using their AP nuclease activity. Specifically, Thermotoga maritima endonuclease IV is a member of the second conserved AP endonuclease family that includes Escherichia coli endonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of the T. maritima endonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of the T. maritima endonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.

Nucleotide Sequence and DNA Secondary Structure, as Well as Replication Protein A, Modulate the Single-stranded Abasic Endonuclease Activity of APE1

A major role of the multifunctional human Ape1 protein is to incise at apurinic/apyrimidinic (AP) sites in DNA via site-specific endonuclease activity. This nuclease function has been well characterized on double-stranded (ds) DNA substrates, where the complementary strand provides a template for subsequent base excision repair events. Recently, Ape1 was found to incise efficiently at AP sites positioned within the single-stranded (ss) regions of various biologically relevant DNA configurations. The studies within indicated that the ss endonuclease activity of Ape1 is poorly active on ss AP site-containing polyadenine or polythymine oligonucleotides, suggesting a requirement for some form of DNA secondary structure for efficient cleavage. Computational, footprinting, and biochemical analyses indicated that the nature of the secondary structure and the proximity of the AP site influence Ape1 incision efficiency significantly. Replication protein A (RPA), the major ssDNA-binding protein in mammalian cells, was found to bind ss AP-DNA with similar affinity as unmodified ssDNA and ds AP-DNA with lower affinity. Consistent with their known relative DNA binding affinities, RPA blocks/inhibits the ss, but not ds, AP endonuclease function of Ape1. Moreover, RPA inactivates Ape1 incision activity at an AP site within the ss region of a fork duplex, but not a transcription-like bubble intermediate. The data herein suggested a model whereby RPA selectively suppresses the nontemplated ss cleavage activity of Ape1 in vivo, particularly at sites of ongoing replication/recombination, by coating the ssDNA.

Cadmium inhibits repair of UV-, methyl methanesulfonate- and N-methyl- N-nitrosourea-induced DNA damage in Chinese hamster ovary cells

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl 2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl- N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl 2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl- N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl 2 the MMS-induced DNA strand breaks accumulated during the first 2 h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl 2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.

Drosophila S3 ribosomal protein accelerates repair of 8-oxoguanine performed by human and mouse cell extracts.

The S3 ribosomal protein of Drosophila melanogaster possesses various DNA repair activities, including the capacity to incise at apurinic/apyrimidinic (AP) sites and 8-oxo-7,8-dihydroguanine (8-oxoG) residues. We have recently hypothesized that this multifunctional protein may improve the efficiency of DNA base excision repair (BER) in mammalian cells. We have investigated the effect of pure GST-tagged Drosophila S3 on BER of different endogenous lesions performed by human and mouse cell extracts. Drosophila S3 significantly accelerated the BER of 8-oxoG (initiated by the bifunctional glycosylase OGG1). The stimulating effect was linked to the capacity of S3 to remove the 8-oxoG lesion and cleave the resulting AP site, rather than acceleration of downstream steps of the BER pathway (e.g., removal of 3' blocking fragments). No stimulating effect was observed on the BER of uracil, natural AP sites, and beta-lyase-cleaved AP sites. Heterologous expression of Drosophila S3 may be used to enhance 8-oxoG repair in human cells.

Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA.

The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. DNA strand cleavage via beta-elimination (beta-lyase) activity coupled with MutY's removal of misincorporated adenine bases was sought using both qualitative and quantitative methods. The qualitative assays demonstrate formation of a Schiff base intermediate which is characteristic of DNA glycosylases catalyzing a concomitant beta-lyase reaction. Borohydride reduction of the Schiff base results in the formation of a covalent DNA-MutY adduct which is easily detected in SDS-PAGE experiments. However, quantitative activity assays which monitor DNA strand scission accompanying base release suggest MutY behaves as a simple monofunctional glycosylase. Treatment with base effects DNA strand cleavage at apurinic/apyrimidinic (AP) sites arising via simple glycosylase activity. The amount of cleaved DNA in MutY reactions treated with base is much greater than that in non-base treated reactions, indicating that AP site generation by MutY is not associated with a concomitant beta-lyase step. As standards, identical assays were performed with a known monofunctional enzyme (uracil DNA glycosylase) and a known bifunctional glycosylase/lyase (FPG), the results of which were used in comparison with those of the MutY experiments. The apparent inconsistency between the data obtained for MutY by the qualitative and quantitative methods underscores the current debate surrounding the catalytic activity of this enzyme, and a detailed explanation of this controversy is proposed. The work presented here lays ground for the identification of specific active site residues responsible for the chemical mechanism of MutY enzyme catalysis.

Protein-Protein Interactions between the Escherichia coli Single-Stranded DNA-Binding Protein and Exonuclease I

It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria.

Excision of Sugar-Phosphate Products at Apurinic/Apyrimidinic Sites by DNA Deoxyribophosphodiesterase of Escherichia coli

It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli. A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate. It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E. coli. The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM. It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione. The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases.

Physical association of the 2,6-diamino-4-hydroxy-5N-formamidopyrimidine-DNA glycosylase of Escherichia coli and an activity nicking DNA at apurinic/apyrimidinic sites.

The 2,6-diamino-4-hydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase of Escherichia coli, which is coded for by the fpg gene, excises purine bases with ring-opened imidazoles. In addition to the DNA glycosylase activity, we report that the Fapy-DNA glycosylase of E. coli has an associated activity, resistant to EDTA, that nicks DNA at apurinic/apyrimidinic (AP) sites. The levels of Fapy-DNA glycosylase and AP-nicking activity were parallel in crude lysates of E. coli HB101 harboring different plasmids constructed from the fpg gene. The fpg gene is different from the xth, nth, and nfo genes of E. coli, whose gene products also cleave DNA at AP sites. The Fapy-DNA glycosylase was purified to electrophoretic homogeneity. During this purification, the Fapy-DNA glycosylase copurified with an AP-nicking activity using chromatographic separations based on ion-exchange, molecular weight exclusion, and hydrophobicity. The cleavage at AP sites by the Fapy-DNA glycosylase left a 5'-phosphomonoester nucleotide at one terminus. In addition, DNA containing reduced AP sites was not nicked by the Fapy-DNA glycosylase. These data suggest that the mechanism of cleavage involved beta elimination. Therefore, this activity of the Fapy-DNA glycosylase nicking DNA at AP sites should be referred to as an AP lyase. The 3' terminus did not prime nick-translation by E. coli DNA polymerase I. However, the 3' terminus becomes a substrate for nick-translation if first allowed to react with calf intestine phosphatase or the E. coli exonuclease III. These data suggest that the repair of the Fapy lesion at least to some extent results in the formation of both 5'- and 3'-phosphomonoester nucleotides and the release of the deoxyribose.

Base excision repair of DNA in gamma-irradiated human cells.

Escherichia coli endonuclease IV was used to incise cellular DNA specifically at apurinic/apyrimidinic (AP) sites prior to alkaline elution to measure the resulting DNA strand breaks. gamma-Irradiated HeLa cells initially contained DNA strand breaks and no AP sites. Upon incubation at 37 degrees C the strand breaks were rapidly repaired and AP sites were generated and subsequently repaired. The transient nature of the AP sites indicates the in vivo operation of a base excision repair pathway whereby damaged bases are removed from DNA by DNA glycosylases to produce AP intermediates that are then substrates for AP endonucleases.