A dot-blot method for quantification of apurinic/apyrimidinic sites in DNA using an avidin plate and liposomes encapsulating a fluorescence dye

A dot-blot method for quantification of apurinic/apyrimidinic (AP) sites in genomic DNA (calf thymus DNA) is described using an avidin-modified glass slip and biotinylated liposomes containing sulforhodamine B as a fluorescence marker. Aldehyde reactive probe (ARP)-tagged DNA was found to be strongly adsorbed on an avidin slip, even if treated with ethanolamine and biotin, with an efficiency of 51% due to the positive surface charge of avidin, and unbound ARP was easily washed out of the surface with Milli-Q water. In the assay protocol, calf thymus DNA containing AP sites is reacted with ARP in solution and immobilized on an ethanolamine- and biotin-treated avidin slip (EAB-avidin slip), followed by incubation with streptavidin. The AP sites were finally quantified with biotinylated liposomes containing 1.5 mM sulforhodamine B as a fluorescence marker. The mean fluorescence intensity over the surface of the slip was an analytically relevant measure of the amount of AP sites in calf thymus DNA. By using the dot-blot assay, 1-5 AP sites per 10(4) nucleotides in 5 and 100 ng of DNA were quantified. The current dot-blot method has potential for quantification of AP sites in genomic DNA at a level of several nanograms.     

Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P-DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 microM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10(6) bp versus 3.7 lesions/10(6) bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3-10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mbeta16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P-DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Detection of abasic sites and oxidative DNA base damage using an ELISA-like assay

Reactive oxygen species produce a wide spectrum of DNA damage, including oxidative base damage and abasic (AP) sites. Many procedures are available for the quantification and detection of base damage and AP sites. However, either these procedures are laborious or the starting materials are difficult to obtain. A biotinylated aldehyde-specific reagent, ARP, has been shown to react specifically with the aldehyde group present in AP sites, resulting in biotin-tagged AP sites in DNA. The biotin-tagged AP sites can then be determined colorimetrically with an ELISA-like assay, using avidin/biotin-conjugated horseradish peroxidase as the indicator enzyme. The ARP assay is thus a simple, rapid, and sensitive method for the detection of AP sites in DNA. Furthermore, removal of damaged base by DNA N-glycosylases generates AP sites that can be measured by the ARP reagent. By coupling the ARP assay with either endonuclease III from Escherichia coli or 8-oxoguanine N-glycosylase (OGG1) from yeast, investigators can rapidly determine the amount of oxidative pyrimidine damage (endonuclease III-sensitive sites) or purine damage (OGG1-sensitive sites) in cellular DNA, respectively. An increased level of oxidative damage has been implicated in several age-related human diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as the aging process. The sensitivity and simplicity of the ARP assay thus make it a valuable method for investigators who are interested in estimating the level of oxidative DNA damage in cells and tissues derived from patients with various age-related diseases or cancers.     

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.     

Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P–DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 μM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10⁶ bp versus 3.7 lesions/10⁶ bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3–10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mβ16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P–DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Properties of a monoclonal antibody for the detection of abasic sites, a common DNA lesion.

The abasic site is one of the most frequent changes occurring in DNA and has been shown to be lethal and mutagenic. An abasic site in DNA can be tagged by reaction with O-4-nitrobenzylhydroxylamine (NBHA), resulting in the formation of an oxime linkage between the abasic site and the NBHA moiety. In order to measure NBHA-tagged abasic sites, a monoclonal antibody was elicited against a 5'-phosphodeoxyribosyl O-4-nitrobenzyl hydroxylamine-BSA conjugate. The antibody was specific for the NBHA residue as demonstrated by hapten inhibition, with IC50 values for 5'-phosphodeoxyribosyl-NBHA, deoxyribosyl-NBHA, ribosyl-NBHA and NBHA of 0.3 microM, 5 microM, 5 microM and 7 microM, respectively. Other haptens examined, including benzylhydroxylamine, 5'-phosphodeoxyribosyl-, deoxyribosyl-, and ribosyl-benzylhydroxylamine, showed no inhibition even at 1 mM. The antibody showed high specificity for NBHA-modified AP sites in DNA and exhibited no cross reactivity with normal DNA bases, otherwise-modified DNA bases or unmodified AP sites. Using a direct ELISA assay, the antibody detected 1 AP site (after NBHA-modification) per 10,000 base-pairs or approximately 10 femtomoles of AP sites in DNA. DNA lesions were detectable in 60Co gamma-irradiated DNA at a dose as low as 10 rad (0.1 Gy) and the production of antibody detectable sites was proportional to the gamma-ray dose. Since NBHA reacts with lesions containing an aldehyde group, the simplicity and sensitivity of the antibody assay should provide a useful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.     

DNA strand scission by antitumor antibiotics. Detection of C-4'-hydroxylated abasic sites in DNA

Detection of C-4'-hydroxylated abasic site in calf thymus DNA was investigated. Upon heating with neutral hydrazine (90 degrees C, 5 min) C-4'-hydroxylated abasic site formed by photo-excited Co(III)bleomycin was converted to the fragments having 3'-phosphoro-3'-pyridazinylmethylate as illustrated on Scheme 1. Subsequent enzymatic digestion of the reaction mixture provided four kinds of pyridazine mononucleotides (1, 2, 3, and 4). The fact that the amount of free bases released from calf thymus DNA corresponded well to the amount of pyridazines indicates this reaction can be used for detection of C-4'-hydroxylated abasic site in DNA. By this assay, we first identified the formation of C-4'-hydroxy abasic sites in calf thymus DNA by neocarzinostatin.     

An assay for RNA oxidation induced abasic sites using the Aldehyde Reactive Probe

There have been several reports describing elevation of oxidized RNA in ageing or age-related diseases, however RNA oxidation has been assessed solely based on 8-hydroxy-guanosine levels. In this study, Aldehyde Reactive Probe (ARP), which was originally developed to detect DNA abasic sites, was used to assess RNA oxidation. It was found that ARP reacted with depurinated tRNA(Phe) or chemically synthesized RNA containing abasic sites quantitatively to as little as 10 fmoles, indicating that abasic RNA is recognized by ARP. RNA oxidized by Fenton-type reactions, γ-irradiation or peroxynitrite increased ARP reactivity dose-dependently, indicating that ARP is capable of monitoring oxidized RNA mediated by reactive oxygen species or reactive nitrogen species. Furthermore, oxidative stress increased levels of ARP reactive RNA in cultured cells. These results indicate the versatility of the assay method for biologically relevant oxidation of RNA. Thus, this study developed a sensitive assay for analysis of oxidized RNA.     

A dominant-negative form of the major human abasic endonuclease enhances cellular sensitivity to laboratory and clinical DNA-damaging agents

Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3' damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu(96) (changed to Gln) and Asp(210) (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue beta-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.     

A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3′-blocking termini following AP lyase cleavage by Nth1.     

An assay for RNA oxidation induced abasic sites using the Aldehyde Reactive Probe

Abstract

There have been several reports describing elevation of oxidized RNA in ageing or age-related diseases, however RNA oxidation has been assessed solely based on 8-hydroxy-guanosine levels. In this study, Aldehyde Reactive Probe (ARP), which was originally developed to detect DNA abasic sites, was used to assess RNA oxidation. It was found that ARP reacted with depurinated tRNA^Phe or chemically synthesized RNA containing abasic sites quantitatively to as little as 10 fmoles, indicating that abasic RNA is recognized by ARP. RNA oxidized by Fenton-type reactions, γ-irradiation or peroxynitrite increased ARP reactivity dose-dependently, indicating that ARP is capable of monitoring oxidized RNA mediated by reactive oxygen species or reactive nitrogen species. Furthermore, oxidative stress increased levels of ARP reactive RNA in cultured cells. These results indicate the versatility of the assay method for biologically relevant oxidation of RNA. Thus, this study developed a sensitive assay for analysis of oxidized RNA.     

Enzymatic release of 5'-terminal deoxyribose phosphate residues from damaged DNA in human cells.

Activities that catalyze or promote the release of 5'-terminal deoxyribose phosphate residues from DNA abasic sites previously incised by an AP endonuclease have been identified in soluble extracts of several human cell lines and calf thymus. Such excision of base-free sugar phosphate residues from apurinic/apyrimidinic sites is expected to be obligatory prior to repair by gap filling and ligation. The most efficient excision function is due to a DNA deoxyribophosphodiesterase similar to the protein found in Escherichia coli. The human enzyme has been partially purified and freed from detectable exonuclease activity. This DNA deoxyribophosphodiesterase is a Mg(2+)-requiring hydrolytic enzyme with an apparent molecular mass of approximately 47 kDa and is located in the cell nucleus. By comparison, the major nuclear 5'----3' exonuclease, DNase IV, is unable to catalyze the release of 5'-terminal deoxyribose phosphate residues as free sugar phosphates but can liberate them at a slow rate as part of small oligonucleotides. Nonenzymatic removal of 5'-terminal deoxyribose phosphate from DNA by beta-elimination promoted by polyamines and basic proteins is a very slow mechanism of release compared to enzymatic hydrolysis. We conclude that a DNA deoxyribophosphodiesterase acts at an intermediate stage between an AP endonuclease and a DNA polymerase during DNA repair at apurinic/apyrimidinc sites in mammalian cells, but several alternative routes also exist for the excision of deoxyribose phosphate residues.     

Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1

Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5' end of the repair gap. When the 5' end is blocked and not amenable to β-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4(-/-) mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species.     

Nuclear DNA endonuclease activities on partially apurinic/apyrimidinic DNA in normal human and xeroderma pigmentosum lymphoblastoid and mouse melanoma cells

DNA endonuclease activities from nuclear proteins of normal human and xeroderma pigmentosum (XP), complementation group A, lymphoblastoid and Cloudman mouse melanoma cells were examined against partially apurinic/apyrimidinic (AP) DNA. Non-histone chromatin-associated and nucleoplasmic proteins, obtained from isolated nuclei, were subfractionated by isoelectric focusing and assayed for DNA endonuclease activity against linear, calf thymus DNA. All of the nine chromatin-associated and three of the nucleoplasmic fractions, which lacked DNA exonuclease activity, were tested for DNA endonuclease activity against both native and partially AP, circular, duplex, supercoiled PM2 DNA. In all three cell lines, four chromatin-associated, but none of the nucleoplasmic fractions, showed increased activity against DNA rendered AP by either heat/acid treatment or by alkylation with methyl methanesulfonate (MMS) followed by heat. One chromatin-associated activity, with pI 9.8, which was not active on native DNA, showed the greatest activity on AP DNA. AP activity was moderately decreased in XP cells and slightly decreased in mouse melanoma cells, as compared with normal cells, in the fraction at pI 9.8. Little or no increased activity was observed in any of the endonucleases from any of the cell lines on MMS alkylated DNA.     

Heat shock protein 70 enhanced deoxyribonucleic acid base excision repair in human leukemic cells after ionizing radiation

Base excision repair (BER) of DNA damage in irradiated THP1 human leukemic cells was stimulated by pretreating the cells with exogenous recombinant Hsp70. The treatment of THP1 cells with recombinant Hsp70 in cell culture promoted repair by reducing the frequency of apurinic, apyrimidinic (AP) sites in DNA before and after 1.3 Gy of radiation. However, by 30 minutes after 2.6 Gy, accelerated repair of abasic sites supervened, which may contribute to the loss of the very-low-dose cell hypersensitivity seen in clonogenic studies of other laboratories. After irradiation with 2.6 Gy, the crucial initial glycosylase step was markedly incomplete when cells had been transfected 24 hours before with a small interfering RNA (siRNA) designed to inhibit synthesis of Hsp70. In confirmation, lysates from irradiated siRNA-treated cells after 2.6 Gy were deficient in uracil glycosylase activity (UDG). Transfection with a scrambled RNA of the same size did not interfere with the glycosylase step, ie, the prompt conversion of damaged pyrimidine sites to abasic sites as well as the subsequent repair of those sites. BER measured by reduction of DNA AP sites before and after low-dose radiation was also deficient in THP1 cells that had been transfected with the siRNA designed to inhibit synthesis of Hsp70. These results implicate BER and the participation of Hsp70 in the repair of DNA in human leukemic cells with the doses of ionizing radiation used in clinical regimens.     

Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease

DNA double-strand breaks (DSBs) are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs) with 3'-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS) can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3' blocked single-strand breaks following the creation of abasic (AP) sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2)-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD) in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3'-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.     

Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite

We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [(14)C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [(14)C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410-416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the (14)C, with a limit of detection of 1 lesion in 10(6) nucleotides in 1 microg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with gamma-radiation and peroxynitrite. For gamma-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified method offers the advantage of providing a straightforward and direct measurement of aldehyde- and ketone-containing strand breaks and abasic sites, with the potential for direct labeling in cells prior to DNA isolation.     

O-(Fluoresceinylmethyl)hydroxylamine (OFMHA): A fluorescent reagent for detection of damaged nucleic acids

5- and 6-O-(Fluoresceinylmethyl)hydroxylamine (OFMHA, 5a, b) were prepared from the corresponding bis-pivaloyl-protected hydroxymethylfluoresceins (1a, b) in 50–70% yield. The hydroxylamine derivatives reacted smoothly with the abasic sites present in acid/heat stressed calf thymus DNA.     

Purification and amino-terminal amino acid sequence of an apurinic/apyrimidinic endonuclease from calf thymus.

An apurinic/apyrimidinic (AP) endonuclease (E.C.3.1.25.2) has been purified 1100 fold to apparent homogeneity from calf thymus by a series of ion exchange, gel filtration and hydrophobic interaction chromatographies. The purified AP endonuclease is a monomeric protein with an apparent molecular weight on SDS-PAGE of 37,000. On gel filtration the protein behaves as a protein of apparent molecular weight 40,000. DNA cleavage by this AP endonuclease is dependent on the presence of AP sites in the DNA. DNA cleavage requires the divalent cation Mg2+ and has a broad pH optimum of 7.5-9.0. Maximal rates of catalysis occur at NaCl or KCl concentrations of 25-50 mM. The amino acid composition and the amino-terminal amino acid sequence for this AP endonuclease are presented. Comparison of the properties of this AP endonuclease purified from calf thymus with the reported properties of the human AP endonuclease purified from HeLa cells or placenta indicate that the properties of such an AP endonuclease are highly conserved in these two mammalian species.     

Mechanism of DNA cleavage and substrate recognition by a bovine apurinic endonuclease

The location of the phosphodiester bond cleaved by homogeneous Mg2+-dependent apurinic endodeoxyribonuclease (EC 3.1.25.2; APE) of bovine calf thymus has been determined by using a 21-mer oligonucleotide containing a single central apurinic site as a substrate. A single product of cleavage consistent with cleavage of the oligonucleotide 5' to the apurinic site, and leaving a 3' hydroxyl group, was identified. This enzyme is, therefore, a class II apurinic endonuclease. The substrate specificities of this enzyme have been determined by using a variety of natural and synthetic DNAs or oligonucleotides containing base-free sites. Calf thymus APE has an absolute requirement for a double-stranded DNA and requires an abasic site as a substrate. The presence of a base fragment such as a urea residue, an alkoxyamine group attached to the C'-1 position of the abasic site, or reduction of the C'-1 aldehyde abolishes the APE activity of this enzyme. Synthetic abasic sites containing either ethylene glycol, propanediol, or tetrahydrofuran interphosphate linkages are excellent substrates for bovine APE. These results indicate that APE has no absolute requirement for either ring-opened or ring-closed deoxyribose moieties in its recognition of DNA-cleavage substrates. The enzyme may interact with the pocket in duplex DNA that results from the base loss or with the altered conformations of the phosphodiester backbone that result from the abasic site.