DNA Damage

Paul W Doetsch, Emory University School of Medicine, Atlanta, Georgia, USA

Various biochemical and physical events alter the structure and properties of DNA and convert it into forms that can have deleterious biological consequences for cells. Technologies exist for the sensitive detection and measurement of DNA damage in cells exposed to various agents.

Secondary article
Article Contents
. Introduction . Spontaneous Alteration of DNA . Environmental Damage to DNA . DNA Damage and Chromatin Structure . Detection and Measurement of Base Damage

Introduction
DNA is the genetic material of all living organisms and therefore its coding information must be passed accurately from one generation to the next. Nevertheless, DNA is quite unstable and has the potential for undergoing a plethora of changes in its base, deoxyribose and phosphate components. The mediators of these changes are errors in the normal cellular nucleic acid transactions of DNA replication, repair and recombination as well as numerous chemicals and spontaneous events from endogenous (cellular origin) and exogenous (environmental origin) sources. The focus of this article will be on the biochemical and physical events that alter the structure and properties of DNA and convert it into forms that can have deleterious biological consequences for cells. Since DNA in eukaryotic cells exists as chromatin (DNA plus associated nuclear proteins) organized into nucleosomal structures with various degrees of compactness, consideration will be given to how DNA damage is thought to aect these structures and, conversely, how DNA organization into higher order structures aects its susceptibility to damaging agents in certain genomic regions over others. In addition, technologies for sensitive detection and measurement of DNA damage in cells exposed to various agents will be discussed. The information provided in this article is important for understanding the biological consequences of genetic damage, which include cell death, mutation and, in certain circumstances, the initial events leading to cancer.

Hydrolysis: deamination and base loss

Water-mediated hydrolysis of the exocyclic amino groups of cytosine, adenine and guanine leads to the production of uracil (Figure 2), hypoxanthine and xanthine, respectively. Of these, deamination of cytosine to uracil is by far the most frequent and is estimated to occur at a rate of approximately 1000 per mammalian cell per day. In contrast, the rates of purine deamination are thought to be about 12% that of cytosine. The high rate of cytosine deamination to uracil makes it a frequent source of spontaneous DNA damage. If left unrepaired, uracil will code for adenine instead of cytosine during DNA replication, resulting in GC to AT transition mutations. Indeed, bacterial cells defective in the major enzyme which removes uracil from DNA show an increased rate of spontaneous mutations of this type. It should be noted that the rate of cytosine deamination in single-stranded DNA is approximately 100-fold higher than that of duplex DNA. The modied DNA base 5-methylcytosine, which is present in a small fraction of DNA in eukaryotic cells but concentrated in regions controlling gene expression in socalled CpG islands, exhibits a higher rate of deamination compared to cytosine and results in conversion to thymine with subsequent generation of a GT mispair. These sites have been found to be mutational hotspots despite the fact that a repair system exists for removal of such GT mismatches. The potential biological consequences of purine deamination products are less well understood; hypoxanthine (arising from adenine) is potentially mutagenic due to its ability to base pair with cytosine. Hydrolysis of the N-glycosidic bond (via base protonation by water) of purines and pyrimidines (Figure 1) leading to the generation of base-free (abasic) sites (Figure 2) is another example of spontaneous DNA damage occurring at high frequency at physiological pH and temperatures. Purines are less stable than pyrimidines and estimates in the order of 10 000 depurinations per mammalian cell per day have been made, with pyrimidine loss being placed at about 1/20th of this rate. Thus base loss at sites of purines and pyrimidines is probably one of the most frequent chemically distinct types of DNA damage in cells. The resulting abasic or AP (for apurinic/apyrimidinic) sites are
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Spontaneous Alteration of DNA

Numerous chemical alterations of DNA can occur in the cell under conditions of normal temperature, pH and metabolic activities. The chemical properties of the base, sugar and phosphate components of DNA are such that numerous sites and reactive centres exist for modication due to spontaneous hydrolysis, oxidation and reactivity with certain types of electrophiles (Figure 1). In addition, the act of DNA synthesis itself during replication can be a source of damage introduced into the genome.

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DNA Damage

* NH2 * N A N N * N*

5'

CH2 H

O H H * NH2 II C N N* O*

P O *

CH2 O H H H O H H3 C T N O * IV * O H H O H III O P O *

* O NH *

P O *

CH2 H

O * N

G N N *

NH* NH2 *

CH2 H

O H H I

O
3'

Figure 1 Sites of chemical modification on DNA. The base (A, T, G, C), deoxyribose and phosphate building block components of DNA are vulnerable to attack by numerous exogenous and endogenous agents. Important examples of DNA modifications at various positions caused by spontaneous hydrolysis (open arrows), particularly at the N-glycosidic bond of G (I) to generate an abasic site or at the exocyclic amino group of U to form uracil (II). Other examples include radical attack by reactive oxygen species (ROS) at several positions (closed arrows) on the deoxyribose and bases such as C-8 of G to produce 8-oxoguanine (III). DNA contains many nucleophilic centres (stars) that are attractive sites for chemical reactions with exogenous and endogenous electrophilic chemicals such as alkylating agents. One example of an important, mutagenic base modification is alkylation at the O6 position of guanine (IV).

noninstructional in that they possess no base-pairing properties and have potential deleterious (toxic and mutagenic) eects unless they are repaired. Abasic sites are somewhat unstable and can also lead to DNA strand breaks.

Oxidative damage
A consequence of normal aerobic metabolism in both prokaryotic and eukaryotic cells is the generation of reactive oxygen species (ROS) in the form of singlet oxygen, hydrogen peroxide (or various organic peroxides),
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superoxide anion radicals and Fenton reaction-generated hydroxyl radicals. Major endogenous sources of ROS in eukaryotic cells include leakage from the mitochondria due to incomplete reduction of O2 to H2O during oxidative phosphorylation, nitric oxide synthesis, peroxisomal metabolism, and phagocytic activity of leucocytes. Many exogenous agents in the cellular environment such as long wavelength ultraviolet light, ionizing radiation, and certain chemicals are also potent sources of ROS. Cells have evolved elaborate defence systems for enzymatically and chemically scavenging ROS but despite these pathways, a substantial oxidative challenge is continuously placed on the genomes of most organisms.

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DNA Damage

5'

O O P O O CH 2 OH O OH H CHO O O H dR O P O O

N N

Uracil

3' Abasic site O H N N O

5'

CH 3

H 3C

O N N H

H O P O O O

H O

3'

Cyclobutane pyrimidine dimer O H N O N N NH NH 2 N N N CH 3

N NH 2

dR

dR

8-Oxoguanine

O6-Methylguanine

Figure 2 Representative DNA-damage products. The five important examples shown here are the abasic site (primarily from spontaneous depurination), uracil (from cytosine deamination), cyclobutane pyrimidine dimer (from ultraviolet light exposure), 8-oxoguanine (from reactive oxygen species), and O6methylguanine (from alkyating agents).

Unlike other classes of DNA-damaging agents that produce one or several related types of base damage, ROS are capable of producing direct DNA strand breaks (via oxygen radical attack of deoxyribose) as well as a multitude of chemically distinct base modications which span the range of potential deleterious biological eects (Figure 1). ROS-generated DNA strand breakage 3 termini contain

phosphates or fragments of the sugar, making them unsuitable for DNA polymerases that require unmodied 3-hydroxyl groups. Some 4060 dierent types of base damage products are produced by ROS, the type and frequency of occurrence being dependent on the specic nature and dose of the ROS-generating agent. In general, these products consist of purine and pyrimidine ring
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DNA Damage

saturation, ring cleavage and ring contraction products as well as exocyclic ring additions and modications. Several of these base damages appear to be major products of a number of dierent ROS-generating agents and include 8oxoguanine, the formamidopyrimidines derived from adenine and guanine, thymine glycol, and 5-hydroxycytosine. 8-Oxoguanine (Figure 2) is highly mutagenic and frequently mispairs with adenine during DNA synthesis either as a damaged template base or as misincorporated 8oxodGMP (from 8-oxodGTP) opposite to template adenines. Estimates for the numbers of oxidative hits inicted on cellular DNA are variable and lie within the range of several hundred to tens of thousands per cell (mammalian) per day. The continuous cellular production of ROS and the subsequent damage inicted upon the genome of cells has led to the notion that oxidative modications are one of the most frequently occurring and pervasive types of DNA damage in nature. In mammalian organisms, including humans, it has been proposed that oxidative DNA damage is directly or indirectly linked to many age-related, degenerative conditions such as neuronal cell death, heart disease and cancer.

Replication errors
During replication, DNA polymerase sequentially adds deoxynucleotide monophosphates (dNMPs) to the growing 3 end of the newly synthesized strand from triphosphate (dNTP) substrates via complementary Watson Crick base pairing with DNA template strand bases. There are several routes by which this mode of DNA synthesis can lead to the generation of damage in the resulting duplex DNA product. AC and GT mispairs can be formed by tautomeric shifts of any of the four DNA bases into a rare structural isomer possessing dierent base-pairing properties. Errors in the DNA polymerase delity resulting primarily from defects in proofreading activity can also contribute to base mismatch formation during DNA synthesis. Other routes include DNA polymerase slippages, miscoding/realignments and template strand dislocations, leading to a variety of potential extra base insertion or deletion events in the newly synthesized strand. Another potential route for introduction of DNA damage during replication is through the incorporation of dNTPs containing base modications such as dUTP and 8-oxodGTP. The fact that organisms have evolved enzymes which specically hydrolyse such altered dNTPs and the observation that mutants lacking such functions have elevated mutation rates support the notion that the DNA synthesis machinery has the potential for incorporating appreciable amounts of base damage through this mechanism.

Methylation
Within the context of a normal cellular environment, DNA bases can be methylated by nonenzymatic or enzymatic routes. Small molecules such as certain nitrosated peptides and polyamines (produced by both bacterial and eukaryotic cells) as well as S-adenosylmethionine are capable of nonenzymatic methylations; the main products of such reactions being 7-methylguanine and 3-methyladenine. Other base methylation products are possible, such as the highly mutagenic O6-methylguanine (Figure 2) but are probably present in extremely small amounts. 7-Methylguanine is relatively nontoxic and is not mutagenic per se, however 3-methyladenine blocks replication and is toxic to cells. It has been estimated that about 600 residues of 3methyladenine are produced per day in a human cell. Both bacterial and mammalian cells possess an enzyme (a methyltransferase) which specically methylates cytosine at the C5 position to produce 5-methylcytosine. Not considered to be DNA damage, 5-methylcytosine mediates important functions in the restriction/modication systems of certain bacteria and in the regulation of gene expression of most eukaryotes. As discussed earlier, the fact that 5-methylcytosine is more prone to deamination (which produces thymine) than cytosine (which produces uracil) has important implications for potential mutational hotspots in the genomes of organisms.

Environmental Damage to DNA

All cells exist in an environment that contains numerous chemical and physical agents which can enter the cell and can cause DNA damage. The modes of action of many toxic agents and the vast majority of known mutagens and carcinogens is via an initial DNA-damaging event.

Ultraviolet (UV) light

Nearly all terrestrial organisms are exposed to solar UV radiation during some part of their life cycles. The solar UV spectrum is divided into three wavelength bands, UV-A (long wavelengths), UV-B (intermediate wavelengths) and UV-C (short wavelengths), which induce dierent subsets of DNA damage products. UV-C is absorbed by the stratospheric ozone layer, leaving UV-A and UV-B as the relevant exposure bands. In humans, exposure to UV-B wavelengths is linked to the development of skin cancer. The most toxic, mutagenic and carcinogenic UV lightinduced DNA photoproducts are cyclobutane pyrimidine dimers (CPDs, Figure 2) and (6-4) pyrimidine-pyrimidone photoproducts (6-4 PPs). These lesions occur between adjacent pyrimidines on the same DNA strand and are the

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DNA Damage

major photoproducts induced by UV-C wavelengths (hence the concern over the loss of the ozone layer) but can also be generated in substantial quantities in chronically exposed cells (e.g. skin cells) by UV-B. Although UVA and UV-B cause the formation of a number of other, less abundant photoproducts such as cytosine photohydrates, their potential for deleterious biological eects is unclear. In humans, defects in the ability to repair CPDs and 6-4 PPs predisposes such individuals to multiple skin cancers at an early age.

Ionizing radiation
Ionizing radiation (IR) from medical X-rays, exposure to nuclear fallout, cosmic rays and naturally occurring sources such as radon causes a diverse spectrum of DNA damage. The major damaging species resulting from IR traversal of a cell are water radiolysis products which include, among several ROS and other radical species, the highly reactive hydroxyl radical. With respect to DNA single-strand breaks and base damage, IR-induced DNA damage mimics that seen for oxidative damage. However, unlike metabolically or chemically generated ROS, IR has the capability to deposit numerous reactive species within the same DNA locale, which leads to the production of multiple, closely spaced damages on both strands of the DNA duplex. Such multiply damaged sites lead to the formation of DNA double-strand breaks (a potentially lethal lesion) as well as clustered base damages and are a considerable challenge for cells to repair.

strands (an interstrand crosslink) via closely opposed guanines. Other bifunctional agents include the anticancer drug cis-diamminedichloroplatinum (II), which modies DNA primarily at sites of two adjacent guanines causing intrastrand crosslinks. It is important to note that many anticancer agents are potent DNA-modication chemicals, whose tumour cell killing eects are probably initiated by the primary DNA damage they cause. An important example of a metabolically activated DNA damaging chemical is benzo[a]pyrene (BPDE). A polycyclic aromatic hydrocarbon, BPDE is a combustion product with broad human exposure occurring through inhalation of polluted air (e.g. automobile exhaust), smoking tobacco and ingestion of food and water contaminated by combustion euents. It is enzymatically converted by elements of the cellular P-450 system to a myriad of products, some of which are potent DNAdamaging agents. BPDE is one of the most potent carcinogenic substances known, with the biologically relevant lesion thought to be a particular guanine adduct. Many other classes of compounds, both natural and synthetic, can be enzymatically activated to species which readily form DNA adducts capable of cell killing or causing mutations.

DNA Damage and Chromatin Structure

In cells, DNA does not exist as a naked, open structure but is tightly associated with proteins and organized into supercoiled, higher order structures to t within the small volume of a bacterial cell or nucleus of a eukaryotic cell. In a eukaryotic nucleus nucleosomes comprise the rst organizational level of chromatin and are further organized into solenoids, nucleolaments and even more highly compact formations leading to the gross structural features of a chromosome. It is reasonable to assume that DNA existing in such structures is not equally accessible at all points along the genome to either chemical or physical agents. The organizational structure of DNA changes according to the levels of activities (e.g. stage of the cell cycle, replication, transcription) in that certain areas may become less compact or dissociate from close associations with proteins and potentially less shielded from damaging agents. Little is known about how the dynamic nature of chromatin structure and its higher organizational levels aects the distribution of damage caused by a particular agent. In the case of UV light-induced DNA photoproducts, the distribution of CPDs (Figure 2) but not 6-4 PPs appears to be aected by DNAhistone interactions. Transcription factor binding to certain DNA regions can, depending on the site examined, result in an increased, decreased or unchanged level of UV photoproduct formation. Certain chemicals are able to inict higher levels of damage to
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Chemicals
Potential cellular exposure in humans to the thousands of naturally occurring and synthetic chemicals in our environment that can damage DNA is thought to be an important determinant in the initial stages of the development of cancer. Chemical exposure by inhalation, contact with skin or through ingestion of foods results in cellular uptake where such compounds are converted nonenzymatically to reactive forms. Alternatively, in the cells attempt to excrete potentially toxic compounds by increasing their water solubility (polarity), some compounds are enzymatically converted to highly reactive molecules (usually electrophiles) capable of reacting with a number of the many nucleophilic centres in duplex DNA (Figure 1). An important class of DNA-damaging chemicals that are nonenzymatically activated are the monofunctional alkylating agents (e.g. methyl methane sulfonate), which can modify DNA at numerous single sites on the bases or phosphate backbone. Such modication produces phosphotriesters and a variety of damaged bases, including the highly mutagenic O6-methylguanine (Figure 2). Bifunctional alkylating agents have two reactive groups and are capable of covalently crosslinking complementary DNA

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single-stranded DNA compared to double-stranded DNA, suggesting that certain replicating segments of the genome containing single-stranded template regions may be particularly vulnerable to modication. Our understanding of the rules governing the inuence of chromatin structure on the potential of DNA for sustaining damage is still at an elementary level.

detection of DNA damage such as that caused by UV light. One advantage of such an immunoassay is the potential for visualizing the location of DNA damage in situ. However, one drawback to the development of sensitive immunological assays for DNA damage is the diculty in producing antibodies specic for a particular type of DNA lesion. Lesions which confer only subtle distortions of duplex DNA structure have proven to be dicult with respect to yielding noncrossreacting antibodies.

Detection and Measurement of Base Damage

The ability to reliably detect DNA damage in various samples ranging from naked DNA to cells exposed to various genotoxic agents is important for understanding the potential biological eects of such agents, including toxicity, mutagenicity and carcinogenicity. A large number of dierent techniques spanning a wide range of sensitivities have been developed to detect and quantify DNA damage induced by radiation and chemicals. Most of these methods involve the utilization of some type of sensitive detection system coupled with a high-resolution separation system including polyacrylamide gel electrophoresis, chromatography and mass spectrometry. This discussion will not be a comprehensive coverage of all techniques currently employed, but will focus on those that show the greatest utility and potential for future use as sensitive detection systems for DNA damage.

Gas chromatography/isotope dilution mass spectrometry

In the past decade, gas chromatography/isotope dilution mass spectrometry (GC/IDMS) has been used to detect and quantify many dierent types of DNA base damage products, primarily those resulting from oxidative and ionizing radiation-induced DNA damage. DNA from either in vitro or cell culture exposures to damaging agents can be simultaneously analysed for multiple damages. This method is dependent on having a suitable set of damaged standards for comparison and is limited by the fact that not all types of damages are stable to the sample preparation (derivatization) procedures and the fact that it is less sensitive compared to several other techniques. Nevertheless, GC/IDMS has found extensive utility for making certain types of DNA-damage measurements, particularly from living cells.

Enzyme probes
Various endo- and exonucleases which either incise DNA at sites of damage (endonucleases) or whose hydrolysis of DNA is blocked by various damages (exonucleases) can be used as specic or nonspecic lesion detectors when used in conjunction with end-labelled, dened sequence segments of sample DNA. Shortened DNA products are then analysed on high-resolution polyacrylamide DNA sequencing-type gels and the damage readout is obtained by analysis of the gel band patterns. This type of method can be employed for the detection and in some cases quantication of both physical and chemical damage both for DNA damaged in vitro and for cells exposed to DNAdamaging agents (Friedberg et al., 1995). One of the advantages of this technique is that it allows the sites of damage to be pinpointed at the level of single-nucleotide resolution. A low-resolution version of this technique employs a simple plasmid nicking assay with the nicked and uncleaved plasmid subpopulations separated from each other by agarose gel electrophoresis.

Polymerase chain reaction-based assays

The polymerase chain reaction (PCR) involves multiple, sequential rounds of DNA synthesis within a dened genomic region and results in the amplication of that region to an analysable DNA product from an original sample containing a relatively small number of molecules. PCR methodologies have been utilized to determine the location and levels of specic types of DNA damage at gene, subgene and nucleotide resolution levels. Quantitative PCR (QPCR) is based on the premise that several DNA lesions are capable of blocking DNA polymerase, resulting in decreased levels of amplication (Yakes and Van Houten, 1997). Ligation-mediated PCR (LMPCR) is a single-sided technique in that the amplied DNA fragments have variable ends on one side (corresponding to the lesion site) and xed ends on the other side (Tornaletti and Pfeifer, 1996). Various DNA repair enzymes that cleave at the sites of specic lesions contained in the original sample allow for site-specic identication and quantication of lesions in single-copy genes at single nucleotide positions. Both techniques have been successfully used to determine the frequency (QPCR and LMPCR) and location (LMPCR) of UV photoproducts and oxidative damage within specic genes of cells damaged with radiation or chemicals. QPCR and LMPCR

Immunoassays
A variety of polyclonal and monoclonal antibodies have been developed for use in various immunoassays for the
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are powerful and sensitive methods that are likely to be used to provide gene-specic damage readouts for many types of genotoxic agents.

References
Friedberg EC, Walker GC and Siede W (1995) DNA Repair and Mutagenesis. Washington DC: ASM Press. Le XC, Xing JZ, Lee J, Leadon SA and Weinfeld M (1998) Inducible repair of thymine glycol detected by an ultrasensitive assay for DNA damage. Science 280: 10661069. Tornaletti S and Pfeifer GP (1996) Ligation-mediated PCR for analysis of UV damage. In: Pfeier GP (ed.) Technologies for Detection of DNA Damage and Mutations. New York: Plenum. Yakes FM and Van Houten B (1997) Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proceedings of the National Academy of Sciences of the USA 94: 514519.

Capillary electrophoresis/laser-induced fluorescence: on the frontier of DNA-damage detection

A recent important advance in the development of DNAdamage detection systems couples immunochemical detection with capillary electrophoresis (CE) and laser-induced uorescence (LIF). CE/LIF exploits the specicity of immunodetection, the high resolving power and small sample requirement for CE and the sensitive detection capabilities of LIF. In the case of detection of thymine glycol, an oxidative and radiation-induced base damage product, CE/LIF is capable of measuring damage at levels of 3 10 2 21 mol (zeptomoles) or a few thousand molecules (Le et al., 1998). This translates to 45 orders of magnitude higher sensitivity than other popular sensitive techniques. One important implication of CE/LIF is that it can detect damage in small cellular samples that have been exposed to low, nontoxic doses of DNA-damaging agents. Thus, it may be possible to use this method in screening human populations for exposures to mutagens and carcinogens or obtaining a background reading of endogenously produced damage in various cell types. It is likely to have great utility for studies monitoring both damage and repair.

Further Reading
Cadet J, Berger M, Douki T and Ravanat JL (1997) Oxidative damage to DNA: formation, measurement and biological signicance. Reviews of Physiology, Biochemistry & Pharmacology 131: 187. Cadet J, Berger M, Douki T et al. (1997) Eects of UV and visible radiation on DNA-nal base damage. Biological Chemistry 378: 1275 1286. Dizdaroglu M (1994) Chemical determination of oxidative DNA damage by gas chromatography-mass spectrometry. Methods in Enzymology 234: 316. Dizdaroglu M and Karakaya A (eds) (1999) Advances in DNA Damage and Repair. New York: Kluwer/Plenum. Friedberg EC, Walker GC and Siede W (1995) DNA Repair and Mutagenesis. Washington DC: ASM Press. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362: 709715. Nickolo JA and Hoekstra MF (eds) (1998) DNA Damage and Repair, Volume 2: DNA Repair in Higher Eukaryotes. Totowa: Humana. Pfeier GP (ed.) (1996) Technologies for Detection of DNA Damage and Mutations. New York: Plenum.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net