DNA Repair Mechanisms

Friedberg

I

REPAIRABLE DAMAGE: IDENTIFICATION AND QUANTIFICATION

REPAIRABLE DAMAGE IN DNA¹

Peter A. Cerutti,     Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610

ABSTRACT

Important contributions to the elucidation of the role of DNA repair for cell viability, mutagenesis, malignant transformation and cell degeneration are expected from experiments in which individual DNA lesions of known chemical structure are investigated. This is important in view of the high degree of complexity of the spectrum of lesions which is induced by most physical and chemical agents by direct action and by Indirect action via the intermediacy of active oxygen species. The formation of thymine damage in baby hamster kidney cells by indirect action was demonstrated for the chemical DNA damaging agents benzo(a)pyrene (B(a)P) and ascorbic acid/Cu²+. It is useful to characterize the biochemical properties of DNA lesions in terms of their excisability, coding properties and regulatory propertiesminor deoxyguanosine-AAF adduct. For the B(a)P-deoxyguanosine adducts these results suggest an inverse relationship between the excisability of the lesions and the mutagenicity and toxicity of the B(a)P-metabolites by which they are induced in mammalian cells. The effect of the location of the lesions in chromatin on their excisability is discussed.

INTRODUCTION

The elucidation of the role of the repair of DNA damage in cell viability, mutagenesis, malignant transformation and cell degeneration represents a major goal in molecular toxicology. Important contributions to this goal are expected from experimental systems in which individual DNA lesions of known chemical structure can be investigated. The study of DNA damaging agents which induce a whole spectrum of lesions and the use of biochemical procedures which do not allow the distinction of individual lesions are less informative. The biochemical and biological effects resulting from the treatment of a cell with a DNA damaging agent are usually a composite of the expression of a multitude of lesions and it is often very difficult or impossible to dissect the contributions made by individual lesions. However, it is the chemical structure of the individual lesions, their abundance and distribution in the genome which determine the mode(s) of lesion processing and ultimately the biological endpoints in a particular cell. Our short range goal should be to define the toxicity, mutagenicity and transforming potency of individual lesions rather than of damaging agents. With the help of this knowledge it may become possible to analyze multiple lesion situations where lesion interaction may influence repairability and biological expression.

In this article I will first discuss lesion formation and point out the complexity of the spectrum of lesions which are formed in the DNA of intact cells by most physical and chemical damaging agents. I will then describe and define biochemical properties of DNA lesions and, as an example, I will review our present knowledge of the relative excisability of a series of structurally related arylation lesions of guanine. In a final paragraph I will discuss the effects which the intragenomic location of lesions may have on their repairability.

(1) FORMATION OF DNA LESIONS BY DIRECT AND INDIRECT ACTION

The list of DNA damaging agents is long and the list of DNA lesions which they produce is even longer. There is hardly an atom on the purine and pyrimidine rings of DNA which has not been shown to react with some physical or chemical damaging agent. Often it is possible to predict the type of lesions which are likely to be formed from basic chemical principles, e.g. a majority of ultimate chemical damaging agents possess electrophilic properties and react with the electron-rich centers of the heterocyclic bases in nucleophilic displacement reactions (1). Valuable information about the structure of DNA lesions can be obtained from in vitro experiments with mono- and polynucleotides but reliable data concerning the relative abundance and distribution of the lesions in situ can only be obtained in experiments with intact cells. Most experiments with chemical agents use highly radioactive compounds in order to be able to detect the introduction of radioactive substituents on the DNA bases (or the backbone) at low levels of damage. However, an important class of DNA lesions escapes detection under these conditions. There are a number of chemical DNA damaging agents of considerable importance to biochemical and medical research which may exert at least part of their action via the formation of reactive oxygen species. Most of these agents, e.g. 4-nitroquinoline-N-oxide (2), streptonigrin(3), adriamycin, mitomycin C(4), benzo(a)pyrene (B(a)P) (5), possess or are metabolically activated to quinone-like structures. In many cell systems they can participate in one-electron redox cycles which lead to the formation of highly reactive oxygen radical species such as superoxide- and hydroxyl-radicals which are expected to induce DNA lesions of the type produced by radiation. Oxygen radical species may also be responsible for the chromosomal damage induced by ascorbic acid plus Cu²+. The formation of DNA strand-breakage by some of these agents has been clearly demonstrated in in vitro component, systems but not in intact cells where drug-induced breaks have to be distinguished from breaks introduced as a consequence of DNA repair.

Hydroxyl- and superoxide- radicals are formed as a consequence of water radiolysis by ionizing radiation. Hydroxyl radicals have been shown to induce thymine damage(8,⁹) and DNA strand breaks(10) in intact cells and they are mostly responsible for the lethal action of aerobic gamma- and x-rays(11). This type of radiation action which is mediated via oxygen radical species is referred to as indirect action. In contrast, energy deposition into the DNA target is referred to as direct action of ionizing radiation. Similarly, active oxygen species are also formed by far-ultraviolet light by water photolysis and by near–ultraviolet light by photodynamic action and photodissociation of hydrogen peroxide(12, ¹³). The type of DNA lesions formed by active oxygen species is expected to be closely related regardless of whether they are formed by ionizing radiation, ultraviolet light or in a redox system involving a chemical agent. Therefore, it may be useful to expand the terminology developed for ionizing radiation to include ultraviolet light and chemical agents and we are proposing the following definitions:

DIRECT ACTION: attack of DNA by the primary agent or a chemical derivative of the primary agent.

INDIRECT ACTION: (of ionizing radiation, ultraviolet radiation or a chemical) attack of DNA by active oxygen species which are formed by the reaction of the primary agent with a non-DNA target.

In general, direct action is expected to predominate for most chemical damaging agents and for far–ultraviolet light while indirect action represents the major intracellular reaction mechanism for ionizing radiation. There is great variation in the structure of DNA lesions induced by direct chemical action. In contrast, a similar spectrum of lesions is expected to be introduced by indirect action of physical and chemical agents. They include: single- and double–strand breaks, products of the 5,6-dihydroxy-dihydrothymine type, damage at the thymine - methyl group, damage involving the other DNA bases, DNA-DNA, DNA–RNA and DNA–protein cross links.

Evidence for indirect action of ultraviolet light and the chemical damaging agents ascorbic acid/Cu²+ and benzo-(a)pyrene (B(a)P) in intact mammalian cells has recently been obtained in our laboratory. The formation of products of the 5,6-dihydroxy-dihydrothymine type (tuv) and the abstraction of tritium from thymine–methyl [³H] was observed in HeLa cells upon irradiation with monochromatic light at 240, 260, 280, 313 and 365 nm. The efficiency of formation of tuv type products was comparable to thymine photodimerization in the near ultraviolet at 313nm(12). There is little doubt that these lesions are formed by indirect action of ultraviolet light. The formation of [³H]H2O in baby hamster kidney cells (BHK) which had been prelabeled in their DNA with thymine–methyl [³H] was observed upon treatment with 2×10−3M ascorbic acid/2×10−5M Cu²+. It was estimated that after 4 hours of incubation approximately 0.2% of all thymine–methyl groups had reacted with hydroxyl-radicals yielding 5-Methylene–uracil radicals and water (M. Ide and P. Cerutti, unpublished). 5-methylene–uracil radicals may in part be repaired chemically to thymine by hydrogen abstraction from neighboring organic molecules and participate in secondary radical chain reactions which may result in DNA strand breakage and DNA cross-linking. BHK cells have retained the capacity in culture to metabolize B(a)P. Poly-cyclic aromatic hydrocarbons are in part metabolized by one-electron oxidation reactions. The 6-oxo–B(a)P–radical represents an important intermediate which is further oxidized to the stable metabolites 6, 12-, 1, 6- and 3, 6-B(a)p–dione. Lorentzen and Ts’o(5) have speculated that the B(a)P-diones might participate in redox cycles and that superoxide radicals, hydrogen peroxide and hydroxyl-radicals might be formed as a consequence. Preliminary results of experiments similar to those described above for the ascorbic acid/Cu²+ system support this hypothesis and suggest the formation of DNA-base damage by indirect action of B(a)P(14).

In summary, the spectrum of lesions induced by most DNA damaging agents is extremely complex. Both physical and chemical agents are capable of producing lesions by direct and indirect action. Lesions induced by indirect chemical action may escape detection when standard experimental protocols are followed. In studies of DNA repair and its relationship to the biological effects of DNA damage it is of greatest importance that agents are chosen which induce a simple spectrum of structurally characterized lesions. The biochemical characterization of individual lesions is discussed in the following section.

(2) BIOCHEMICAL PROPERTIES OF DNA LESIONS

The biological expression of a lesion is mediated by its biochemical properties which in turn are determined by its chemical properties. The chemical structure of individual lesions is, of course, independent of the cell system in which they are being studied but their biochemical properties and biological expression are not. Therefore, questions concerning lesion chemistry can often be adequately studied in vitro but the biochemical and biological properties of lesions have to be investigated in the intact cell. Major chemical properties of lesions are their electronic configuration, hydrogen bonding potential, stereochemistry and solvation and their effect on local DNA- and chromatin-conformation. Major biochemical properties include excisability, persistence, coding properties and regulatory properties. It may be useful to describe these terms for our present discussion.

EXCISABILITY: Efficiency by which a lesion is removed from the genome in a particular cell. Excisability is expressed as the fraction (and amount in μmoles lesion per mole DNA-phosphate) of a specific lesion removed from the genome in one generation under non-saturating conditions. Under non-saturating conditions the fraction of the lesions removed per time unit is independent of the initial lesion concentration. Several pathways, such as nucleotide-excision and base-excision, contribute to total excisability and it may be possible to assess their relative contributions. The structural characteristics of a lesion which determines its excisability by individual excision pathways are expected to be different. Excisability may vary for different portions of the genome and for chromatin subfractions such as the DNAse I or the staphylococcal nuclease digestible and resistant portions of chromosomal DNA (see below).

LESION PERSISTENCE: The fraction and amount of a specific lesion which remains in the genome after the excision rate has dropped below detectibility.

CODING PROPERTIES: The chemical structure of lesions which are present in the genome when they are reached by the replication- and transcription- machinery determines whether they are instructive or non-instructive(15). Instructive lesions may code normally, i.e. possess the coding properties of the parent nucleotide from which they are derived or they may miscode. Non-instructive lesions have lost the ability to act as a template and may or may not act as a temporary or permanent block to replication and/or transcription. The presence of non-instructive lesions may result in the perturbation of daughter strand synthesis and in perturbation of transcription.

REGULATORY PROPERTIES: DNA lesions per se or, DNA configurations formed as intermediates during lesion processing, may induce or modulate DNA-repair and DNA-synthesis functions. It may be necessary in the future to define additional biochemical properties of lesions, e.g. repairability by mechanisms which do not involve lesion excision, efficiency for the induction of sister chromatid exchanges and chromosomal aberrations, etc.

Ideally the biochemical properties of prototype DNA lesions should be defined in standard pro- and eu-karyotic cell systems in quantitative rather than qualitative terms since the situation will be rare where a lesion is not excised at all, miscodes a 100% of the time, is completely non-instructive, etc. Prokaryotic systems, in particular E. coli and its bacteriophages, are extremely useful for quantitative studies of this type. Analogous experiments with mammalian viruses are promising and have already yielded important results. The characterization of the biochemical properties of DNA lesions in intact mammalian cells is more difficult. However, progress is being made in the determination of the relative excisability and persistence of individual, chemically characterized lesions in intact mammalian cells and some of this work is summarized below.

Excisability is a raw measure of the efficiency of lesion removal in a particular cell which is defined because more precise enzymological terms cannot be applied to the intact cell. The term does not contain information about the mechanism(s) of lesion excision or the subsequent steps of repair which accomplish reconstitution of the DNA strand. Excisability is measured by observing the disappearance of specific lesions or specific endonuclease sensitive sites from high molecular weight DNA and cannot, in general, be assessed by repair replication phenomena. The term is biologically meaningful if lesion removal is rate determining and if different excision repair pathways possess comparable fidelity. Excisability is determined by lesion properties and cellular properties. Important lesion properties are: chemical structure, effect on local DNA and chromatin conformation(16), intragenomic location (see below); Important cellular properties are: tissue and species, state of growth, state of differentiation.

The excisability of several structurally characterized lesions has been assessed in pro- and eukaryotic cells. I have chosen recent work with human cells as an example for our discussion. A simple relationship between the excisability of (presumably) pyrimidine photodimers and the toxicity and mutagenicity of far-ultraviolet light was observed by Maher and her collaborators (17, ¹⁸) for the different complementation groups of Xeroderma pigmentosum (XP). An inverse relationship was found between residual excisability of photodimers and mutagenicity and lethality of equal doses of ultraviolet light. In analgous experiments the toxicity and mutagenicity of K–region epoxides of several polycyclic aromatic hydrocarbons and of N–acetoxy–AAF were found to be highest in the XP–strains with lowest residual excisability of far-ultraviolet lesions. However, the excisability of the lesions introduced by these ultimate chemical damaging agents was not determined and no direct correlation is possible, therefore. A similar relationship may exist between the sensitivity of Ataxia-telangiectasia skin fibroblasts and their capability to remove Micrococcus luteus γ-endonuclease sensitive sites(19) but no quantitative correlation has been possible so far.

In our laboratory we are investigating the relative excisability of prototype guanine lesions in human epitheloid lung cells A549(20) with the aim to relate excisability and toxicity and to define the structural properties of the lesions by which excisability is determined. This cell line is particularly useful since it has retained the capability to metabolize drugs such as polycyclic aromatic hydrocarbons in culture. The following arylation lesions of guanine have been studied: (1) The major products formed upon exposure of A549 to benzo(a)pyrene (B(a)P)(21) (7R) N²-(10-{7β, 8α, 9α–trihydroxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene}deoxyguanosine (B(a)P-diol-epoxide I–dG) and (7R or 7S) N²-(10-{7β, −8α, 9β-trihydroxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrenelyl)deoxyguanosine (B(a)P-diol-epoxide II-dG) (2) the guanine and possibly cytosine adducts formed upon exposure of A549 to the K-region epoxide 4,5-dihydrobenzo(a)pyrene-4,5-epoxide (B(a)P-4, 5-epoxide C1 and B(a)P-4,5-epoxide C2) (3) the lesions formed upon reaction of A549 with N-acetoxy-AAF: major lesion 8-[N-(aminofluorenyl)]deoxyguanosine (AAF-(C8)dG and its deacetylated derivative 8-[N-(aminofluorenyl)] deoxyguanosine (AF- (C8)dG); minor lesion N²-[3-(2-acetyl-aminofluorenyl)]deoxyguanosine (AAF-(N2)dG)(22). In all these guanine lesions bulky aryl-substituent have been introduced but the position of substitution and the size, stereochemistry and electronic properties of the polycyclic ring systems are different. In a first series of experiments each damaging agent was studied separately and in a second series pairs of damaging agents were studied in the same culture. Under the latter conditions effects of toxicity and cell growth on excisability are the same for the lesions which are being compared and lesion interaction can be investigated. The results of a typical experiment with B(a)P plus N-acetoxy-AAF are given in Figure 1 and 2. Figure 1 shows the chromatographic profiles of DNA digests of A549 cultures which were first treated with [³H]B(a)P for 48 hours, then with [¹⁴C]-N-acetoxy-AAF for 45 min. and subsequently incubated for 0 and 21 hours. The initial levels of damage and the amount of each individual lesion removed as a function of post-treatment incubation is plotted in Figure 2 (it was not possible to measure DNA-acetylation in our experiment). From experiments of this type the following scale of excisability of arylation lesions of guanine was derived: (The values listed above the lesions represent the fractions of lesions removed within one generation time under non-saturating conditions. Relevant data from other investigators obtained with different human cells is included for comparison (23–²⁵)).

Fig. 1 Sephadex LH-20 chromatography of enzymatic digests of DNA from A549 cells which were prelabeled with ³²PO−24 grown for 48 hrs. in the presence of 1.3μM [³H]benzo(a)pyrene and then treated with 6μM [¹⁴C]N-acetoxy-acetylaminofluorene and incubated for 0 or 21 hrs. Ratios of the ¹⁴C and ³H radioactivity in each fraction over total ³²P radioactivity contained in the DNA digest are plotted in order to correct for DNA damage dilution due to cell growth. A1, minor AAF lesion, AAF-(N2)dG; A2, major AAF lesion AAF-(C8)dG; A3 (probably) deacetylated major AAF lesion AF-(C8)dG; B1, B(a)P-diol-epoxide I-dG; B2, B(a)P-diol-epoxide II-dG.

Fig. 2 Kinetics of excision from DNA of benzo(a)pyrene and N-acetoxy-acetylaminofluorene lesions in A549 cells. Data was derived from experiments of the type shown in Fig. 1 and the same symbols are used for the lesions. The horizontal lines mark the level of the individual lesions at the outset of post-treatment incubation. Left side: B(a)P lesions (lesion B0 has not been chemically identified); right side: AAF lesions.

It is interesting to note that there appears to exist on a qualitative level an inverse relationship between excisability of the B(a)P lesions and mutagenicity (26–²⁸) and carcinogenicity (29) in rodent systems of the ultimate damaging agents by which they are induced. The lowest excisability was found for the minor product introduced by N-acetoxy-AAF, i.e. AAF-(N2)dG, which has been shown to be persistent in rat liver DNA. The effect of the intragenomic distribution of the individual lesions on their excisability is being investigated (see below.)

It will be important to relate excisability to lesion structure. We have proposed earlier to distinguish classes of DNA lesions on the basis of the magnitude of their effect on the local conformation of the DNA helix.(16) In recent experiments the relative single strandedness of DNA containing the arylation products of guanine discussed above was determined by comparing the susceptibility of the damaged DNA to digestion by single-strand specific S1-nuclease (30–³²). In general it appears that lesions inducing extensive local single-strandedness also possessed relatively high excisability in A549 cells. These findings point out the potential usefulness of a classification of DNA lesions on the basis of local helix distortion(16) but they do not prove that helix distortion is the only or the most important lesion property which determines excisability.

(3) EFFECT OF DNA AND CHROMATIN STRUCTURE ON LESION FORMATION AND REPAIRABILITY

Numerous factors are expected to influence the chemical reactivity and the repairability of individual residues in DNA in situ in a living cell. They include (1) DNA structure: primary sequence, regional stability and conformation of the DNA helix. The effect of DNA structure on its chemical reactivity has been studied in detail for the case of pyrimidine photodimerization and this topic has been subject of recent reviews(33). Little is known about the effect of DNA structure on the formation of lesions by chemical damaging agents except that residues in single stranded regions are usually more reactive than residues in double stranded regions. (2) Replicative and transcriptional activity: DNA in the process of replication or transcription may differ in its chemical reactivity and repairability from inactive DNA. Evidence for increased repair activity in DNA growing points relative to bulk DNA has been obtained in human lymphoblastoid cells following treatment with N–methyl–N′-nitro–N–nitrosoguanidine and methylnitrosourea(34). Preferential introduction of 2-acetyl-aminofluorene (AAF) into the DNA portions which were highly sensitive to pancreatic DNAse I (at 8-10% solubilization) was observed in rat liver within 10 to 30 min. following intraperetoneal injection of the drug but not at later times(35). On the basis of results obtained in the reticulocyte and oviduct system(36, ³⁷) it can be speculated that transcriptionally active genes were damaged at a higher rate by AAF than the rest of the DNA immediately after application of the drug. (3). Nuclear structure: DNA located on the nuclear periphery in contact with the nuclear membrane may differ in reactivity and repairability from DNA located towards the center of the nucleus. (4) Chromatin structure: DNA located in the nucleosomal core particle may differ in its reactivity and repairability from linker DNA; further reactivity differences may originate from the interaction with individual histones and non-histone proteins. The DNA repair field rapidly exploits progress made in basic molecular biology. It will be interesting to employ any new technology which allows the separation of structurally or functionally distinct chromatin regions for the study of damage formation and repair.

Recent progress in the understanding of chromatin structure and in particular the development of simple procedures which distinguish between nucleosomal-core and -linker DNA on the basis of their resistance and susceptibility to digestion by staphylococcal nuclease allow a first look at the effect of chromatin structure on lesion distribution and repairability. In experiments with highly reactive ultimate chemical damaging agents the nucleosomal linker DNA was more reactive than core DNA. The damage level of linker DNA was approximately twice that of core DNA upon treatment of isolated chromatin from duck erythrocytes with N-acetoxy-AAF(38) The adduct content of linker DNA was 4 times and 11 times higher for the major two lesions formed by the reaction of intact human alveolar tumor cells A549 with benzo(a)pyrene-4,5-epoxide (G. Feldman and P. Cerutti, unpublished). Higher susceptibility of linker DNA may also be found for damage introduced by indirect action of chemical and physical agents since the nucleosomal proteins may act as efficient quenchers of the reactive oxygen species which are formed as intermediates.

Evidence for more rapid repair of lesions in linker DNA has been obtained in human skin fibroblasts following ultraviolet irradiation. Immediately after irradiation repair replication occurred preferentially in staphylococcal nuclease digestible linker DNA(39, ⁴⁰). Similar results were obtained following treatment of mouse mammary gland with methylmethanesulfonate(41). A higher initial level of damage in the linker DNA may in part be responsible for these results, especially for the studies using methylmethanesulfonate. Evidence for non-random distribution of alkylation damage has been obtained earlier(34)

The relative rate of the following processes influence the intragenomic distribution of the lesions for damaging agents which require metabolic activation (1) rate of formation of ultimate damaging agent(s) (2) relative rates of lesion formation and removal in different parts of chromatin. The rates of these processes and therefore the level of lesions and their distribution in DNA are expected to undergo continued changes following administration of the drug. Experiments with the liver procarcinogens dimethylnitrosamine (DMN) and AAF are in agreement with this expectation. The intragenomic distribution of methylated bases and AAF-adducts and their removal from DNA was measured in rat liver. In both cases the damage level was considerably higher in the nuclease digestible portions of chromatin soon after drug treatment. The damage levels decreased and became more comparable in the digestible and resistant DNA fractions when the post-treatment period was extended over several days(35, ⁴²). A similar result was obtained when human alveolar tumor cells A549 were treated with N-acetoxy-AAF (J. Remsen and P. Cerutti, unpublished). Since staphylococcal nuclease was used in the studies with AAF it can be concluded that the formation and removal of AAF-adducts was more efficient in the nucleosomal linker DNA. No attempt was made to distinguish individual lesions in this work. However, there is no reason to expect equal patterns of distribution for all lesions introduced by a given damaging agent. This question is of particular importance since the excisability and persistence of individual lesions are likely to be influenced by their location in the genome. Equal distribution of B(a)P-diol-epoxide II-dG between nucleosomal core- and linker- DNA was observed in actively B(a)P metabolizing A549 cells while the level of B(a)P-diol-epoxide I-dG was slightly higher in the core-DNA. The excisability of the two adducts was the same in core- and linker DNA during post-treatment incubation up to 34 hours (J. Remsen, G. Feldman and P. Cerutti, unpublished).

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¹This work was supported by contract EY-76-S-05-4155 of the U.S. Department of Energy and P.H.S. Grant GM18617.

Workshop Summary: DISTRIBUTION AND QUANTITATION OF DNA DAMAGE

Ann Ganesan,     Department of Biological Sciences, Stanford University, Stanford, California 94305

Publisher Summary

One of the most persistent problems encountered in the study of DNA repair has been the quantification of various DNA lesions at levels that are of biological significance. Most chemical and even radiochemical procedures are not sufficiently sensitive. This chapter discusses the information that has been complicated by the fact that most DNA damaging agents—UV, X-radiation, alkylating agents—produce several types of lesions and in different proportions. It is desirable to know precisely what type of lesion the enzyme recognizes to use an enzyme for measuring DNA damage. The chapter explains the difficulties encountered in determining the type of lesion recognized by an enzyme when the substrate DNA contains more than one kind of damage and the enzyme preparation being examined contains more than one activity.

One of the most persistent problems encountered in the study of DNA repair has been the quantification of various DNA lesions at levels which are of biological significance. Most chemical and even radiochemical procedures are not sufficiently sensitive. Recently, assays using specific enzymes have been developed which show the necessary sensitivity. However, the accuracy of these assays depends upon knowing what type of damage is recognized by the enzyme used. Obtaining this information has been complicated by the fact that most DNA damaging agents – UV, X-radiation, alkylating agents, for example – produce several types of lesions, and in different proportions. The disparity in the sensitivity of chemical and enzymatic assays often makes it difficult to identify which of the lesions an enzyme considers its proper substrate. The situation is further complicated by the apparent diversity of enzymes with overlapping but non-identical specificities. In spite of these difficulties, however, the sensitivity and relative simplicity of enzymatic assays is resulting in increasingly wider use (see 1 for review).

The prototype lesion for developing the enzymatic assays was the pyrimidine dimer. Two endonucleases specific for dimers, one from Micrococcus luteus and one from bacteriophage T4, have been used to determine the pyrimidine dimer content of UV-irradiated DNA. The assays measure the number of nicks per DNA molecule resulting from endonuclease treatment. Their sensitivity, therefore, depends upon the molecular weight of the untreated control DNA. Typically this has been 1−2 × 10⁸ in studies of DNA repair in bacterial or mammalian cells. With DNA of this size the number of pyrimidine dimers produced by 1−2 J/m² at 254 nm can easily be measured in a sample of 100 ng of DNA. This corresponds to approximately 2 dimers per 10⁸ daltons, or 10−17 μmoles of pyrimidine dimers per 10⁶ DNA phosphates. Recently developed methods which permit measuring 1−2 nicks per 10⁹ (2, 3, 4) daltons should increase the sensitivity of the assays by a factor of 10.

Two procedures have generally been used to quantify nicks for these assays. Linear DNA has been sedimented in alkaline sucrose gradients and the number of nicks produced by endonuclease treatment calculated from number average molecular weights (5). Alternatively, in vitro studies have often utilized superhelical DNA as substrate. In this case the conversion of supercoils to nicked circles has been determined using sedimentation, electrophoresis or binding to nitrocellulose filters to separate nicked from unnicked molecules. The average number of nicks per molecule has been calculated by the Poisson expression from the proportion of molecules remaining unnicked. The procedures should give similar results although the underlying principles are different. However, data presented in this workshop indicated that the apparent yield of nicks produced by the pyrimidine dimer specific T4 endonuclease V as a function of UV dose to the substrate DNA was two-fold higher for superhelical than for linear DNA (Ganesan, Friedberg and Seawell, unpublished results). The discrepancy appeared to be due to the method of analysis, not to a difference in the response of superhelical and linear DNA to UV irradiation or to T4 endonuclease V. This conclusion was based upon studies of ColE1 DNA treated with the restriction endonuclease EcoRI to produce unit length linear molecules. When analysed on alkaline sucrose gradients UV irradiated DNA treated with T4 endonuclease V appeared to contain the same number of nicks whether EcoRI treatment occurred before irradiation, after irradiation or after T4 endonuclease V treatment. The cause of the discrepancy is not known, but may reflect a slightly non-random distribution of pyrimidine dimers in irradiated DNA (cf. Grossman, this volume).

Enzymatic assays have primarily been used to detect pyrimidine dimers but they can, in principle, be extended to any lesion for which a specific endonuclease or DNA glycosylase can be found. Although glycosylases do not nick DNA they produce apurinic or apyrimidinic (AP) sites which will yield nicks upon treatment with alkali or AP endonuclease. Some enzymes of actual or potential interest for detecting damage include AP endonucleases (6, 7), 3-methyl adenine glycosylase (8), uracil glycosylase (9, 10) and endonucleases active against unknown lesions in DNA treated with γ- or X-rays (11; Wallace, et al., this volume), osmium tetroxide (12) or acetylaminofluorene (13).

In order to use an enzyme for measuring DNA damage it is desirable to know precisely what type of lesion the enzyme recognizes. Two of the speakers in this workshop drew attention to difficulties encountered in determining the type of lesion recognized by an enzyme when the substrate DNA contains more than one kind of damage and the enzyme preparation being examined contains more than one activity. These difficulties were exemplified by experiments with enzymes which attack alkali labile sites in DNA exposed to alkylating agents or ionizing radiation (Brent, et al., this volume; Wallace, et al., this volume). Alkali labile sites in this context are lesions which will yield DNA strand breaks under alkaline but not under neutral conditions. Two types have recently been distinguished: 1) those sites sensitive to treatment with 0.1 N NaOH for 20 minutes at 37°C; 2) those requiring more extensive treatment, e.g. 4 hours at room temperature in 1 M glycine-NaOH (pH 12.8). According to Teebor, et al (this volume) AP sites belong to the first category, and acid heat depurinated DNA contains only this type of site. However, DNA exposed to X-radiation in the presence of KI, alkylated with MMS or irradiated with high doses of UV contain both types. They are not AP sites, but they may be altered bases which can dissociate to leave AP sites. 3-methyl adenine may belong in this category. Currently the only way to distinguish the two types of alkali labile site is to specify the treatment required to convert them to strand breaks. The distinction is important for characterizing enzyme preparations. Purified AP endonucleases attack only the first type of site. However, other enzyme preparations will attack both types. In some cases this results from the presence of more than one enzyme (8; Brent, et al., this volume), while in other cases a single endonuclease appears to be responsible (Wallace, et al., this volume).

Several laboratories are currently purifying and characterizing enzymes involved in DNA repair. As knowledge of individual biochemical reactions contributing to repair increases so will the number of enzymes available for measuring DNA damage and the variety of lesions which can be measured enzymatically. At the present time, however, the characterization of enzymes and the identification of lesions produced by various physical and chemical agents are interdependent investigations. Frequently the first evidence for an erstwhile unrecognized enzyme is its effect upon a previously unsuspected lesion. Since many of the enzyme preparations now being used to monitor DNA damage are not highly purified and contain activities other than the one of primary interest they may yield exaggerated estimates of a particular type of damage. It is essential to remember this limitation when interpreting results of enzymatic assays.

Illustrating a different approach to quantifying DNA damage Rahn, et al. (this volume) described a model system using iodinated cytosine. DNA containing 5-iodo cytosine (IoCyt) can be produced in vivo by growing bacteria with IoCyt, and in vitro by a chemical reaction of iodide with single stranded DNA (14, 15). Irradiation of DNA containing iodinated bases with 313 nm light causes selective loss of iodine, which can be monitored using ¹²⁵I. Two potentially interesting applications of this system were indicated. 1) The types of lesion produced by photolysis should be limited in number and therefore relatively easy to characterize and quantify; 2) the chemical iodination reaction strongly prefers cytosine in single stranded DNA (15) and may provide a probe for damage caused by agents which act on guanine and disrupt hydrogen bonds.

References

1. Paterson, M.C. Adv. Radiat. Biol. 1977; 7. [in press].

2. Ahnström, G., Erixon, K. Int. J. Radiat. Biol. 1973; 23:285–289.

3. Sheridan, R.B., Huang, P.C. Nucleic Acids Res. 1977; 4:299–318.

4. Kohn, K.W., Friedman, C.A., Ewig, R.A.G., Iqbal, Z.M. Biochemistry. 1974; 13:4134–4139.

5. Rupp, W.D., Howard-Flanders, P. J. Molec. Biol. 1968; 31:291–305.

6. Verly, W.G., Rassart, E. J. Biol. Chem. 1975; 250:8214–8219.

7. Ljungquist, S. J. Biol. Chem. 1977; 252:2808–2814.

8. Laval, J. Nature. 1977; 269:829–831.

9. Lindahl, T., Ljungquist, S., Siegert, W., Nyberg, B., Sperens, B. J. Biol. Chem. 1977; 252:3286–3294.

10. Tye, B.-K., Chien, J., Lehman, I.R., Duncan, B.K., Warner, H.R. Proc. Nat. Acad. Sci., U.S. 1978; 75:233–237.

11. Paterson, M.C., Smith, B.P., Lohman, P.H.M., Anderson, A.K., Fishman, L. Nature. 1976; 260:444–447.

12. Gates, F.N., Linn, S. J. Biol. Chem. 1977; 252:2802–2807.

13. Van Lancker, J.L., Tomura, T. Biochim. Biophys. Acta. 1974; 353:99–114.

14. Chan, H.-C., Ruyechan, W.T., Wetmur, J.G. Biochemistry. 1976; 15:5487–5490.

15. Commorford, S.L. Biochemistry. 1971; 10:1993–1999.

LESIONS IN ALKYLATED DNA DETERMINED BY SUSCEPTIBILITY TO ALKALI, APURINIC ENDONUCLEASE OR N-GLYCOSIDASE¹

Thomas P. Brent*, George W. Teebor** and Nahum J. Duker+,     *Dept. of Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN 38101; **Dept. of Pathology, New York Univ. Medical Center, New York, NY 10016; +Dept. of Pathology and Fels Research Inst., Temple Univ. Health Sciences Center, Philadelphia, PA 19140

ABSTRACT

To detect and quantitate lesions in methyl methanesulfonate-treated DNA, we converted these sites to strand breaks by 5 different parallel procedures. Apurinic sites alone were so converted by apurinic endonuclease from human CEM-CCRF lymphoblasts or by a 20-min treatment with 0.1 M NaOH. Lesions in addition to these were converted by whole-cell extracts (CEM-CCRF or HeLa), by apurinic endonuclease plus 3-methyladenine N-glycosidase (both from CEM-CCRF), or by a 4-hr treatment with 1.0 M glycine-NaOH.

INTRODUCTION

The simplest way to determine physically or chemically induced lesions in DNA is to degrade the DNA and isolate the altered moiety. When this is not feasible, primary lesions may be converted to more readily measured secondary forms. In the case of methyl methanesulfonate-treated DNA (MMS-DNA), enzyme activities from human cells convert some damage to strand breaks and may be useful probes for specific lesions. However, reports of the susceptibility of MMS-DNA to damage-specific enzymes from human cells are in apparent conflict.

Duker and Teebor (1) reported that maximum strand breakage following treatment with HeLa-cell sonicates was equal to that following alkaline treatment by the method of Lindahl and Andersson (2). Because alkali-labile sites, determined by the latter method, had been equated with apurinic sites in assays with heat-depurinated DNA (2), the conclusion was that human-cell extract acted only on apurinic sites in MMS-DNA. By contrast, Brent (3) reported that lesions in addition to apurinic sites were sensitive to N-glycosidase activity, isolated from CEM-CCRF lymphoblasts. In this study, however, the methods for quantitating apurinic sites differed from those used by Duker and Teebor. Hence, discrepancies between the studies might be related to the specificity of the procedures used to determine apurinic sites. We now report a comparative study of procedures, including the ones used in these two studies, for determining alkali-labile and enzyme-sensitive sites in MMS-DNA.

METHODS

DNA Treatments.

PM2 viral DNA was either heat-depurinated at 70°C, pH 5.5 (4), or alkylated with MMS (12.5×10−3M) at 37°C for 5, 10, or 30 min and re-isolated by gel filtration (1).

Assays of Alkali-Labile Sites.

Method A: DNA was incubated in 1.0 M glycine-NaOH for 4 hr at room temperature (2). Method B: DNA was incubated in 0.1 M NaOH for 20 min at 37°C (5, 6). In both methods, the prevalence of strand breaks was estimated from the proportion of nicked DNA, determined by alkaline-CsCl sedimentation analysis (4).

Assays of Enzyme-Sensitive Sites.

Alkylated PM2 DNA was diluted to 0.3–0.5 μg/100 μl in 0.5 mM dithiothreitol, 0.06 M Hepes-KOH (pH 8.0), and 0.6 mM MgCl2 prior to addition of 5 μl of apurinic endonuclease purified from human CEM-CCRF lymphoblasts (5). In some assays, 3 μl of N-glycosidase from CEM-CCRF cells was also added (3).

For assays with crude-cell extracts, 20 μl of HeLa-cell sonicate or 10 μl of CEM-CCRF cell homogenate (in 0.01 M Tris-HCl, pH 8.0, 1 mM EDTA and 0.5 mM dithiothreitol) was added to 10 μl of 1.0 M Tris-phosphate (pH 7.0), 1 mM EDTA. The final reaction volume was 120 μl.

All enzyme reactions were for 30 min at 37°C. The extent of strand breakage was estimated from the proportion of nicked DNA, determined by neutral-CsCl sedimentation analysis (4). Background values (for strand breaks in DNA prior to assay) have been subtracted for all reported data.

RESULTS AND DISCUSSION

When heat-depurinated PM2 DNA was treated with alkali according to methods A or B, comparable numbers of strand breaks were produced in each case (7). Consistent with previous reports (4, 5, 8), these values also corresponded closely with the number of apurinic sites converted to strand breaks by either crude extract from HeLa cells or by purified apurinic endonuclease from CEM-CCRF lymphoblasts.

By contrast to these results for heat-depurinated DNA discrepancies occurred among results for these same methods when MMS-DNA was analyzed. Table 1 shows that alkaline hydro lysis by method A gave about three times as many strand breaks as did method B. Assays with purified apurinic endonuclease, a specific probe for apurinic sites, gave values comparable to those of method B for alkali-labile sites. We have estimated from the data reported by Ljungquist et al. (9) that their MMS-DNA also had about 3 alkali-labile sites (determined by method A) for every 1 site that was sensitive to purified apurinic endonuclease from calf thymus—consistent with our findings.

TABLE 1

ALKALI- AND ENZYME-SENSITIVE SITES IN MMS-TREATED DNA

aPurified apurinic endonuclease.

bFrom HeLa cells (*) or CEM-CCRF cells (**).

cPurified apurinic endonuclease combined with partially purified N-glycosidase.

dEach value is the average number of strand breaks per DNA molecule.

Our interpretation of these data is that method B allows the accurate determination of apurinic sites that exist immediately after MMS treatment, while method A appears to promote the hydrolysis of additional sites, possibly alkylated bases or phosphotriesters (6). The contention that alkylated DNA is stable at high pH (9) does not appear to hold for the conditions of method A.

In further comparisons, the amount of strand-breakage caused by crude extracts was over twice that by purified apurinic endonuclease (Table 1). However, with the addition of N-glycosidase to apurinic endonuclease, strand breakage approached that of crude extracts. These findings suggest that crude extract attacks more than one kind of lesion: apurinic sites (sensitive to endonuclease) and alkylated bases (sensitive to N-glycosidase plus endonuclease). Supporting evidence for this includes a recent report by Laval (10) that 3-methyladenine N-glycosidase combined with apurinic endonuclease (both purified from M. luteus) produces strand breakage at non-apurinic sites in MMS-DNA. Similar to the bacterial enzyme, the human N-glycosidase that we used releases 3-methyladenine but not 7-methylguanine, and thus, may be a suitable probe for the former in alkylated DNA.

In conclusion, our findings emphasize the need to establish the specificity and stoichiometry of various methods for converting primary lesions to secondary forms. While all of the procedures that we assessed are quantitative for apurinic sites in heat-depurinated DNA, only those done with purified apurinic endonuclease or alkaline hydrolysis by the methods of Brent (5) or Shooter and Merrifield (6) are specific for these sites in MMS-DNA. Whole-cell extracts, on the other hand, provide a measure of the sum total of enzyme-sensitive damage, while the Lindahl-Andersson method of alkaline hydrolysis (2) may detect a broader spectrum of damage.

REFERENCES

1. Duker, N.J., Teebor, G.W. Proc. Nat. Acad. Sci., U.S.A. 1976; 73:2629.

2. Lindahl, T., Andersson, A. Biochem. 1972; 11:3618.

3. Brent, T.P. Nucleic Acids Res. 1977; 7:2445.

4. Teebor, G.W., Duker, N.J. Nature. 1975; 258:544.

5. Brent, T.P. Biochim. Biophys. Acta. 1976; 454:172.

6. Shooter, K.V., Merrifield, R.K. Chem. Biol. Interact. 1976; 13:223.

7. Teebor, G.W., Goldstein, M.S., Duker, N.J., Brent, T.P.Hanawalt P.C., Freidberg E.C., eds. DNA Repair Mechanisms. Academic Press, 1978. [(in press)].

8. Ljungquist, S., Lindahl, T. J. Biol. Chem. 1974; 249:1530.

9. Ljungquist, S., Andersson, A., Lindahl, T. J. Biol. Chem. 1974; 249:1536.

10. Laval, J. Nature. 1977; 269:829.

¹This work was supported by USPHS Grants CA-14799, CA-16669, and RR-05584, and by ALSAC.

ENZYMATIC RECOGNITION OF DNA DAMAGES INDUCED BY IONIZING RADIATION¹

Susan S. Wallace, Paul R. Armel and Harold L. Katcher,     Department of Microbiology, New York Medical College, Valhalla, New York 10595

ABSTRACT

Both alkali-labile and alkali-stable damages in x-irradiated PM2 DNA are recognized by purified fractions of the Escherichia coli x-ray endonculease. An activity, partially purified from Saccharomyces cerevisiae using an x-irradiated PM2 DNA substrate, appears only to recognize alkali-labile lesions.

INTRODUCTION

An enzyme activity partially purified from E. coli on the basis of its ability to nick x-irradiated PM2 DNA (1, 2), has been shown to recognize alkali-labile and alkali-stable damages in such DNA (2–4), as well as DNA damages induced by osmium tetroxide (3). A similar activity has also been demonstrated in Micrococcus luteus extracts (5), and has recently been resolved into five fractions (6). Apurinic endonucleases isolated from E. coli (7, 8), and other sources (for a review see 9), also nick x-irradiated DNA presumably at apurinic/apyrimidinic sites.

In this study, we report the specificity of purified fractions of the E. coli x-ray endonuclease as well that of partially purified Saccharomyces cerevisiae (yeast) apurinic activities.

RESULTS AND DISCUSSION

The E. coli x-ray endonuclease has been further purified by DNA agarose affinity chromatography and phosphocellulose ion exchange chromatography. In 10 mM Tris, pH8, the enzyme elutes at 0. 6M KCl on DNA agarose; in 10 mM MKPO4, pH 6.5 it elutes at 0.5 M KCl on phosphocellulose. By using these methods, 5000 fold purification is achieved with small scale preparations, while 1000–2000 fold purification is achieved with larger-scale preparations. Fraction IV exhibits the same specificity on x-irradiated PM2 DNA substrate as does less purified fractions or crude extracts. That is, with DNA x-irradiated in the presence of the hydroxyl radical scavanger potassium iodide (KI), the enzyme recognizes fast-converting alkali-labile lesions (204), slow converting alkali-labile lesions (4), and alkali-stable damages (2–4). Strand breaks, whether radiation-induced, alkali-induced, or enzyme-induced, are measured by the conversion of PM2 Type I DNA to Type II using sucrose gradient sedimentation analysis (1–4). E. coli x-ray endonuclease Fraction IV also nicks osmium tetroxide-treated DNA, heat depurinated DNA and ultraviolet-irradiated DNA containing 180 pyrimidine dimers per PM2 molecule. Purified fractions do not exhibit any activity on unirradiated DNA either in the presence or absence of magnesium; and after DNA-agarose chromatography, it is not necessary to add calf thymus DNA to compete for non-specific nucleases. As with crude preparations, purified E. coli x-ray endonuclease does not require magnesium, has a broad pH optimum (6.5–8.5) and is fully active in the presence of 10mM