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Chapter 2 : DNA Damage
Category: Microbial Genetics and Molecular Biology
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This chapter deals with different types of DNA damage, and introduces general principles of DNA damage recognition. For convenience, DNA damage can be divided into two major classes, referred to here as endogenous and environmental. The “endogenous” category includes mainly hydrolytic and oxidative reactions that are a consequence of life surrounded by water and reactive oxygen. The “environmental” class includes physical and chemical agents that cause DNA damage, often generated outside cells. While all of the primary components of DNA (bases, sugars, and phosphodiester linkages) are subject to damage, much of the chapter focuses on the nitrogenous bases, since these specify the genetic code. The importance of chromatin is emphasized in the context of damage to DNA. The influence of chromatin structure and protein binding on the distribution of DNA damage has bearing on the responses of living cells to damage, since there is evidence that sites of base damage in chromatin are not equally accessible to DNA repair enzymes. The chapter considers the structural features of damaged DNA that can be specifically recognized by proteins to initiate DNA repair.
Major sites of hydrolytic and oxidative damage in DNA. A short segment of one DNA strand is shown with the four principal DNA bases. The major sites of hydrolytic depurination are shown by long solid gold arrows. Short solid gold arrows show other sites of hydrolytic attack. Major sites of oxidative damage are indicated by the dotted gold arrows. (Adapted from reference 294 .)
Products formed from the deamination of bases in DNA. The standard numbering of the base ring atoms is indicated.
Deamination of cytosine to uracil (U) and of adenine to hypoxanthine (HX) can result in base pair transitions. The U and HX pair as T and G, respectively, during semiconservative DNA synthesis. The top panel of the figure shows a replicating DNA molecule in which U and HX have already mispaired. A second round of DNA replication is just beginning. As this second replication fork proceeds (lower panel), replication of the template strand containing the A and C results in transition (see chapter 3) of the G-C and T-A base pairs to A-T and C-G base pairs, respectively.
Proposed mechanisms for the hydrolytic deamination of cytidine to uridine ( 447 ). Path → III → IV is analogous to the hydrolysis of an amide. It is called the direct route and involves direct attack at the 4-position of the pyrimidine ring by a hydroxyl ion. Loss of ammonia yields uridine. Path I → II → V → is called the addition-elimination mechanism and involves addition of water to the 5 ,6 double bond of protonated cytidine to yield cytidine hydrate (dihydrocytidine) (II). Further attack by water is followed by the loss of ammonia, yielding uridine hydrate (dihydrouridine) (V), which is dehydrated to uridine (IV). In DNA, similar reactions can occur, where R symbolizes the deoxyribose-phosphate backbone. (Adapted from reference 447 .)
Mechanism of deamination of cytidine by bisulfite ( 447 ). Cytidine is converted to a sulfonated derivative (5,6-dihydrocytidine-6-sulfonate), which is then hydrolytically deaminated at acidic pH to yield a sulfonated uridine derivative (5,6-dihydrouridine-6-sulfonate). At alkaline pH, this derivative is converted to uridine. (Adapted from reference 447 .)
Uracil can be incorporated into DNA from dUTP during semiconservative DNA synthesis. The dUTP pool is generated both from dCTP and from dUDP. In wild-type cells the pool size of dUTP is small relative to that of dTTP, since most dUTP is degraded to dUMP by dUTPase. The dCTP deaminase in many bacteria, including E. coli, is a major source of dUTP, while yeast, animal cells, and some other bacteria instead have a dCMP deaminase that generates dUMP. (Adapted from reference 260 .)
The formation of thymidylate from dUMP is catalyzed by the enzyme thymidylate synthetase. During this reaction, 5,10-methylenetetrahydrofolate is converted into dihydro-folate and regeneration of tetrahydrofolate is catalyzed by dihydrofolate reductase. Inhibition of dihydrofolate reductase by amethopterin (methotrexate) results in reduced levels of tetrahydrofolate and hence reduced conversion of dUMP to dTMP. (Adapted from reference 260 .)
Dependence of the logarithms of the rate constants (k) (reciprocal seconds) on pH and H0 (a parameter used to indicate acidity of pH < 1) at 95°C, for deoxyribonucleoside hydrolysis. At acidic pH, depurination occurs more rapidly than does depyrimidination. (Adapted from reference 447 .)
Mechanism of strand breakage in DNA by β-elimination. Deoxyribose residues at sites of base loss exist in equilibrium between the open (aldehyde) form shown in the figure and the closed furanose form (not shown). In the aldehyde form, 3’-phosphodiester bonds are readily hydrolyzed by a β-elimination reaction in which the pentose carbon beta to the aldehyde is activated at alkaline pH, as shown.
Cellular reactions leading to oxidative damage of DNA via the Fenton reaction. H2O2 is formed by endogenous metabolism or is available exogenously. Superoxide is produced as a byproduct of O2 reduction in the electron transport chain. Superoxide dismutation and release of protein-bound iron by superoxide form H2O2 and Fe2+, respectively, which in turn can react to form •OH-type oxidants. These oxidants may cause DNA damage. Fe3+ produced by the Fenton reaction may be reduced by available NADH, thus replenishing Fe2+. H2O2 can be depleted by peroxidases, peroxiredoxins, and catalase, which utilize reduced glutathione, thioredoxin, cytochrome c, ascorbate, etc. (Adapted from reference 197 .)
Formation of thymine glycol by •OH radical attack at thymine. (Adapted from reference 455 .)
Imidazole ring opening in adenine and guanine following radical attack can yield the FaPy products shown. (Adapted from C. J. Chetsanga, M. Lozon, C. Makaroff, and L. Savage, Biochemistry 20:5201-5207, 1981.)
Scheme showing 7,8-dihydro-8-oxoguanine (8-oxoG) mispairing with adenine in DNA. The 8-oxoG is shown in the syn conformation, having rotated from the anti conformation about the bond indicated by the arrow. ( 50 .)
A major product of lipid peroxidation is malondialdeyde (MDA), which reacts with G, A, and C bases in DNA to form the M1G, M1A, and M1C adducts shown. (Adapted from reference 319 with permission of Oxford University Press.)
A major product of lipid peroxidation is 4-hydroxynonenal, which can give rise to the exocyclic etheno adducts of A, C, and G in DNA. The epoxide, 2,3-epoxy-4-hydroxynonanal, reacts with DNA bases to form an intermediate that can lead to etheno adducts as shown. (Adapted from reference 319 .)
Examples of DNA base damage induced by ionizing radiation and other agents that generate reactive oxygen species. 8-Hydroxyguanine occurs more commonly in the isomeric form 7,8-dihydro-8-oxoguanine ( Fig. 2–13 ).
Average annual effective-dose equivalent of ionizing radiation to a member of the population in the United States. Data from reference 82 .
Examples of damage induced by ionizing radiation to the sugar-phosphate backbone moieties.
(A) The UV radiation spectrum. (Adapted from Friedberg, et al., [ 144 ] with permission.) (B) Comparison of the average absorption spectrum for affecting DNA and the Sun’s spectrum at the Earth’s surface. Many action spectra for the biological effects of UV radiation coincide with the DNA absorption spectrum ( 443 ). The solar spectrum at the Earth’s surface was calculated for Gainesville, Fla. for 2.3 mm ozone and a zenith angle of 25°. (Adapted from reference 442 .)
Illustration of a CPD and a (6–4)PP in DNA, both shown as examples of photoproducts at a TC sequence.
Structure of cis-syn and trans-syn cyclobutane thymine T<>T dimers and their approximate three-dimensional orientations in B-form DNA. (Adapted from reference 486 and D. Weinblum and H. Johns, Biochim. Biophys. Acta 114:450-59, 1966.)
Determining the location of CPD in UV-irradiated DNA by using a specific enzyme probe. A double-stranded DNA fragment (only one strand is shown) is radiolabeled at the 5’ ends and incubated with saturating amounts of an enzyme such as T4 denV (see chapter 6), which specifically recognizes CPD in DNA. The enzyme cuts the 5’-glycosyl bond of the dimer and also the 3’ -phosphodiester bond as shown. This procedure leaves stable 5’ -end-labeled DNA fragments with lengths that bear a precise relationship to the sites of CPD. The DNA is then loaded onto a denaturing polyacrylamide sequencing gel which includes additional DNA-sequencing reaction samples as size markers ( 170 ). (Adapted from reference 170 .)
Dose-response curves for cyclobutane T<>T dimer formation in a DNA fragment of known nucleotide sequence. The different curves show the dose response for individual dimer sites, which are identified numerically by their location in the sequenced DNA by the numbers in parentheses ( 170 ). The CPD were quantified from the amount of radioactivity present in bands separated by electrophoresis by the technique shown in Fig. 2–22 . (Adapted from reference 170 .)
Percentage of incision at (6–4)PP by hot-alkali treatment of simian virus 40 DNA following exposure to increasing doses of 254-nm UV-C radiation ( 45 ). The average positions of incisions at several different sites are shown for each dose and each dinucleotide class of photoproduct. (Adapted from reference 45 .)
Formation of 5-thyminyl-5,6-dihydrothymine (spore photoproduct) by the addition of two different radicals of thymine generated by UV radiation. (Adapted from K. C. Smith, p. 67-77, in M. A. Pathak, L. C. Harber, M. Seiji, and A. Kukita [ed.], Sunlight and Man, University of Tokyo Press, Tokyo, Japan, 1974.)
Example of a monomeric pyrimidine base lesion caused by UV radiation.
The absorption of a photon can promote an electron to one of several short-lived excited states termed singlet states, which are characterized by antiparallel electron spins. Return to the ground state by photon emission is accompanied by fluorescence. However, spin inversion results in the longer-lived triplet state, which can facilitate further reactions. The energy levels are shown for the lowest excited singlet states (S1) and lowest triplet states (T1) of adenine (A), guanine (G), cytosine (C), and thymine (T), along with that of acetophenone φAc). The lowest triplet energy state of <J>Ac is slightly higher than that of thymine but lower than that of the other DNA bases. Thus, on irradiation of DNA at about 300 nm, the triplet energy of <<Ac is transferred to thymine, thereby facilitating the formation of CPD between adjacent thymines. (6–4) photoproducts are not formed via a triplet state intermediate. (Adapted from A. Lamola, p. 17-55, in M. A. Pathak, L. C. Harber, M. Seiji, and A. Kukita [ed.], Sunlight and Man, University of Tokyo Press, Tokyo, Japan, 1974.)
Chemical structures of several representative simple alkylating agents that react with DNA.
Chemical structures of alkylating chemotherapeutic agents that react with DNA. (Adapted from reference 281 .)
Nucleophilic centers in DNA that are the most highly reactive with alkylating agents. In general, the ring nitrogens of the bases are more reactive than the ring oxygens. Alkylations at phosphodiester linkages (to yield phosphotriesters), N7 of guanine, and N3 of adenine are the most frequently encountered.
Representation of interstrand DNA cross-linking by nitrogen mustard through the N7 positions of two guanine bases on opposite strands of a duplex.
Cisplatin can form intrastrand adducts in DNA, monoadducts, interstrand DNA cross-links, protein-DNA crosslinks, and glutathione-DNA cross-links. (Adapted from reference 115 .)
Detection of interstrand DNA cross-links by isopyncnic sedimentation. DNA uniformly labeled with [14C]thymidine ([14dTC]) is replicated in the presence of [3H]BrdU to generate DNA of intermediate density, in which one strand is light and the other is heavy. In the absence of cross-linking (left), denaturation of the DNA and sedimentation in alkaline cesium chloride yield 3H (heavy, H) and 14C (light, L) peaks of radioactivity. However, when the two DNA strands are cross-linked (right), DNA of intermediate density (HL) results.
Structures of psoralen and some psoralen derivatives.
Intercalation of psoralen with DNA to form two types of monoadducts (A and B) or a diadduct (interstrand DNA cross-link) (C). Two types of monoadducts can result because the 5,6 double bond of thymine can photoreact with psoralen at either its 3,4 double bond or its 4’,5 double bond (see Fig 2-34). The formation of the cross-link requires independent UV absorption events at each reactive end.
Projection of psoralen (A) and angelicin (B) molecules intercalated between two base pairs in DNA. In each case the thymines shown are on opposite strands of the DNA duplex. Note that angelicin cannot cross-link two DNA strands, because one end of the molecule has an angular configuration that is not appropriately juxtaposed with one of the thymines.
Scheme for the metabolic activation of nonpolar polycyclic chemicals by the cytochrome P-450 system in a mammalian cell to form reactive intermediates that bind to nucleophilic centers in DNA, ER, endoplasmic reticulum. (Adapted from D. W. Nebert, M. Negishi, L. W. Enquist, and D. C. Swan, p. 351-362, in C. C. Harris and P. A. Cerutti [ed.], Mechanism of Chemical Carcinogenesis, Alan R. Liss, Inc., New York, N.Y., 1982.)
Metabolic activation of AAF proceeds through the formation of N-hydroxy intermediates before the formation of N-acetoxy-AAF and other esterified forms. These compounds are highly reactive with the C8 (left and middle) and to a lesser extent the N2 (right) positions of guanine in DNA. (Adapted from reference 333 .)
Metabolic products of the metabolism of benzo[a] pyrene by microsomal mixed-function oxygenases. Some of the products are electrophilic epoxides that have high reactivity for nucleophilic centers in DNA. The 7,8-diol-9,10-epoxide is thought to be the ultimate carcinogenic form of benzo[a]pyrene. (Adapted from J. K. Selkirk, M. C. Macleod, C. J. Moore, B. K. Mansfield, A. Nikbakht, and K. Dearstone, p. 331-349, in C. C. Harris and P. A. Cerutti [ed.], Mechanism of Chemical Carcinogenesis, Alan R. Liss, Inc., New York, N.Y., 1982.)
Chemical structure of aflatoxin B1.
Metabolic activation and detoxification pathways for aflatoxin B1. Cytochrome P-450 isoenzymes metabolize aflatoxin B1 ( Fig. 2–41 ) to the 8,9-epoxide, which can react with DNA. Alternatively, detoxification may take place via an epoxide hydrase or conjugation to glutathione. While a number of products are formed, the initial major adduct forms from reaction of the aflatoxin B1 epoxide with the N7 position of guanine in DNA. This adduct has a destabilized glycosyl bond and can depurinate to form an AP site. Alternatively, the primary adduct can undergo opening of its imidazole ring, giving rise to the chemically and biologically stable formamidopyrimidine adduct, aflatoxin B1-FaPy. (Adapted from J. D. Groopman and L. G. Cain, p. 373-407, in C. S. Cooper and P. L. Grover [ed.], Chemical Carcinogenesis and Mutagenesis I, Springer-Verlag KG, Berlin, Germany, 1990.)
Formation of a strong electrophilic agent by reaction of N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) with a cysteine residue, typically part of glutathione. (Adapted from reference 280 .)
Primary structure of bleomycins. The left-hand portion of the molecule is involved in metal chelation, and the shaded portion is important in DNA binding. (Adapted from reference 392 .)
Chemistry and geometry of bistranded lesions induced by bleomycin and enediyne DNA-cleaving agents. Arrows indicate the nucleotides attacked in prominent or consensus cleavage sites, and numbers indicate the particular carbon attacked in deoxyribose. In cases where cleavage in one of the strands is substantially more efficient, the stronger attack site is shown by a solid arrow and the weaker site is shown by an open arrow. For calicheamicin, only the strongest of several target sequences is shown. (Adapted from reference 392 .)
Strand break formation by topoisomerase inhibitors. Topoisomerases bind to DNA and form transient cleavage complexes involving covalent linkage of topoisomerase to DNA ends. In these complexes, topoisomerases I and II form single- and double-strand DNA breaks, respectively, to enable strand passage in the topoisomerase reaction. In the presence of topoisomerase inhibitors (poisons), levels of cleavage complexes (shown in brackets) increase dramatically. Collision of a DNA replication fork with such a complex results in double-strand and single-strand breaks in DNA. (Adapted from reference 145 .)
The Haemophilus ducreyi CDT holotoxin consists of three subunits, a DNase I-like nuclease (CdtB) bound by two ricin-like lectin domains (CdtA and CdtC). (Adapted from reference 351 .)
The nucleosome structure influences the probability of UV radiation-induced CPD formation. Nucleosome fibers are isolated, and linker DNA is digested with micrococcal nuclease. The remaining nucleosomal core DNA is 5’-end labeled and subjected to digestion with the 3’ → 5’ exonuclease activity of T4 DNA polymerase. The exonuclease digestion arrests at positions of photoproducts, and the resulting mixture of end-labeled fragments is separated in a polyacrylamide gel. Untreated DNA is completely digested. The band intensity reflects the probability of photoproduct formation at a defined distance from the nucleosomal core border (however, it does not provide information about sequence preferences, since each nucleosomal core contains a different DNA sequence). If the chromatin fibers and not the naked DNA were irradiated with UV, the photoproduct distribution shows a 10.3-bp periodicity. This periodicity is largely abolished if the CPD are selectively removed by treatment with CPD photolyase before exonuclease digestion.
The hydrogen-bonding groups of DNA bases provide a pattern of sequence-specific binding interactions that is “read” by proteins. The DNA code of hydrogen-bonding interactions was first proposed by Rich and coworkers ( 435 ). These interactions include hydrogen bond donors (black semicircles), and hydrogen bond acceptors (black troughs). The C5 methyl group of thymine (white oval) is frequently contacted by a hydrophobic residue(s) of DNA-binding proteins. The major groove surface of double-stranded DNA presents a much richer syntax of interactions than the minor groove.
An α-helix fits snugly into the major groove of DNA, where it can make sequence-specific interactions with the edges of the base pairs. The amino acid side chains of the basic region from the leucine zipper transcription factor GCN4 interact with complementary groups of the binding site. (Adapted from reference 120 .)
Small drug-like molecules such as netropsin bind in the minor groove of DNA with specificity for patterns of AT-rich and GC-rich regions. The minor groove displays the “universal” hydrogen bond acceptor groups (O2 of pyrimidines and N3 of purines), as well as the 2-amino group of guanine for interaction with small-molecule and protein ligands (see Fig. 2–50 ). (Adapted from reference 258 .)
Crystal structures of the phage λ repressor protein in complex with DNA operator sites were among the first to reveal sequence-specific interactions of proteins with DNA. The λ repressor dimer binds to adjacent major-groove surfaces on one side of the DNA, inserting a helix-turn-helix motif into the major groove for sequence-specific interactions. (Adapted from reference 190 .)
The repressor protein of the lambdoid phage 434 binds to a DNA site with an AT-rich sequence at the center. The highly propeller-twisted A-T base pairs strongly influence the DNA binding affinity of the 434 repressor, even though these base pairs are not directly contacted by the protein ( 4 ). A series of hydrogen bonds between contiguous base pairs in the propeller-twisted conformation contributes to the rigidity of this sequence. This example shows how the local “stiffness” of the DNA can indirectly but significantly affect binding by proteins.
A general pathway for the excision repair of DNA damage could include the formation of an initial encounter complex (step A), followed by distortion of the DNA to expose a damaged nucleotide (step B) and insertion of the substrate into the enzyme active site (step C). Following the enzymatic reaction to excise the damage (step D), the resulting product complex dissociates (step E). Catalytic selectivity for damaged DNA could arise from enhanced exposure of damaged (versus normal) nucleotides (step B), specific binding of damaged substrates in the active site (step C), or a higher rate of the chemical reaction in complex with damaged nucleotides (step D) relative to undamaged DNA.
Base flipping is a common strategy used by DNA repair enzymes for exposing nucleotides in double-stranded DNA to gain access to the active site ( 416 ). A ribbon diagram of human 3-methyladenine DNA glycosylase (gold) is shown engaging a flipped out 1,N 6-ethenoadenine (εdA) in DNA. Tyrosine 162 inserts in the minor groove, stabilizing the εdA nucleotide in an extrahelical conformation. The strong distortion of the DNA in the enzyme complexes and the high rate of base flipping that has been measured for some enzymes suggest that base flipping is an active process and does not result from capture of bases that are spontaneously exposed during the normal breathing of DNA base pairs. (Adapted from reference 473 .)