Endogenous DNA damage and mutation

214

Review

TRENDS in Genetics Vol.17 No.4 April 2001

Endogenous DNA damage and mutation Lawrence J. Marnett and John P. Plastaras In humans, approximately 107 cells divide per second. Estimates suggest that spontaneous mutations arise in about a third of those cells. These mutations arise as mistakes in DNA replication and when DNA polymerases copy damaged templates. The latter result from chemical hydrolysis of nucleoside bases or by reaction of DNA with electrophiles or reactive free radicals generated during metabolism (endogenous DNA damaging agents). This article highlights recent discoveries and emerging opportunities in the study of endogenous DNA damage and mutation.

DNA damage is an important cause of genetic disease. The chemical events that lead to DNA damage include hydrolysis, oxidation and electrophilic attack. These reactions are triggered by exposure of cells to exogenous chemicals (e.g. environmental agents, food constituents, etc.), or they can result from endogenous metabolic processes. Exogenous chemicals were once thought to be the major sources of DNA damage in human beings, but advances in analytical chemistry reveal the existence of diverse and abundant types of damage resulting from endogenous sources. The integration of synthetic chemistry with molecular biology provides strategies for the construction of small genomes containing single lesions at defined sites for evaluation of mutagenic potential and repair. And the generation of organisms bearing targeted deletions in specific DNA repair genes makes it possible to evaluate the mutational consequences of increased steady-state levels of particular types of lesions. The study of endogenous DNA damage is expanding rapidly and space precludes a comprehensive treatment of the field1–6. However, several recent studies provide insight into the causes, mechanisms and consequences of this background damage. We will highlight these studies and offer some opinions of where the field of endogenous DNA damage could be headed.

Nakamura and Swenberg used this approach to quantify AP sites in tissue DNA9. They found levels corresponding to 50 000-200 000 AP sites per genome in many human and rodent tissues. This is unexpectedly higher than the 9000 AP sites per genome per day calculated from the rate of chemical hydrolysis of DNA10. Characterization of the AP sites present in tissues indicates they are cleaved 5′ to the AP site but are poorly repaired by the dRp-lyase activity of DNA polymerase β (Ref. 11). This suggests they are not typical hydrolytic derivatives of AP sites (Fig. 2; Ref. 12). This unique type of AP site might arise by oxygen radical attack on the sugar phosphate backbone of DNA. Typical AP sites induce basepair substitutions (primarily AP site → T)13,14, but the 5′-cleaved AP sites might also induce frameshift mutations such as those detected in microsatellite sequences following treatment of plasmids with H2O2 (Ref. 15). It will be interesting to identify the structure of these poorly repaired 5′-cleaved AP sites. Atamna et al. used a similar approach to detect AP sites in intact cells. Treatment of cultured cells or isolated leukocytes with 3 mM ARP reagent for up to 60 minutes does not reduce cell viability and traps in situ those AP sites that arise spontaneously or as a result of glycosylase-catalyzed repair16. Atamna et al. estimate a steady-state level of AP sites of <0.67 per 106 nucleotides (~4000 per genome), which is substantially lower than the levels measured in DNA isolated from tissue11. The levels of AP sites are significantly increased by treatment of cells with oxidizing agents or methylating agents, which reflects their formation during glycosylase-catalyzed repair16. Interestingly, repair is significantly reduced in high-passage cell lines compared with low-passage cell lines or leukocytes from older donors, reflecting an age-dependent decline in base-excision repair.

Apurinic/apyrimidinic sites L.J. Marnett J.P. Plastaras A.B. Hancock Jr Memorial Laboratory for Cancer Research, VanderbiltIngram Cancer Center, Center in Molecular Toxicology, Dept of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. *e-mail: [email protected]. vanderbilt.edu

Apurinic/apyrimidinic (AP) sites can be produced by spontaneous hydrolysis, alkylation-induced hydrolysis or glycosylase-catalyzed base-excision repair (Fig. 1). AP sites are rapidly repaired in cells by 5′-phosphodiester hydrolysis (by AP endonuclease) followed by 3′-phosphate elimination (by the dRp-lyase activity of DNA polymerase β) (Fig. 2; Ref. 7). Transiently formed AP sites can be analyzed by trapping with a biotinylated hydroxylamine reagent (the ARP reagent) followed by immunochemical quantification of the oxime product using avidinconjugated horseradish peroxidase (Fig. 2, Ref. 8).

Exocyclic adducts

Oxygen radical attack on DNA leads to a plethora of oxidized bases, as well as strand scission. The products of base oxidation have been extensively studied and much information is available on their generation, mutagenic potential and repair17–22. This extensive literature will not be reviewed because of space limitations. Rather, we will focus on other adducts formed indirectly as a result of other cellular oxidative processes. Polyunsaturated fatty acids are abundant constituents of phospholipid membranes and are

http://tig.trends.com 0168–9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02239-9

Review

TRENDS in Genetics Vol.17 No.4 April 2001

Mutation Apoptosis

Metabolism, inflammation Adducts Electrophiles, free radicals Repair Glycosylase

DNA

Strand breaks

Repair Spontaneous AP sites

Apoptosis

Mutation TRENDS in Genetics

Fig. 1. Sources and consequences of endogenous DNA damage. Aberrant replication of pristine or damaged templates gives rise to mutations. Blockade of replication at damage sites can lead to the induction of strand breaks or to apoptosis. The spontaneous mutation rate in humans is estimated to be 5 × 10–11 mutations per base per cell division89.

highly prone to oxidation; they represent the major targets of oxygen radical attack in cells23. Recent attention has focused on exocyclic DNA adducts that are formed from bifunctional electrophiles generated from the oxidation of polyunsaturated fatty acids (Fig. 3; Ref. 24). Exocyclic adducts are of interest because they block the Watson–Crick base-pairing region and are anticipated to be highly mutagenic. However, some exocyclic adducts exhibit relatively low mutagenicity because they undergo hydrolytic ring-opening to form acyclic adducts that do not block base-pairing25–32. In addition, significant differences have been observed in the mutagenicity of the same exocyclic adduct measured in bacterial and mammalian cells27. Exocyclic etheno adducts (denoted by ε; i.e. εA, εC and N2,3-εG; Fig. 4) are present in many human tissues, and their levels increase in clinical situations associated with enhanced production of oxygen radicals33. Detectable levels of the chemically related etheno adduct, N2,3-εG are present in rodent tissues and can be increased by oxidative stress34. The precise chemistry that leads to their formation is uncertain, but they appear to be derived from oxidized polyunsaturated fatty acids. Some studies suggest that the adducts might arise from the epoxy derivative of 4-hydroxynonenal (Fig. 3; Refs 35,36). The levels of εA and εC are elevated in patients with Wilson’s disease and hemochromatosis, disorders resulting in hepatic accumulation of copper and iron, respectively37. Accumulation of metal ions leads to increased rates of unsaturated fatty acid oxidation. http://tig.trends.com

215

Elevated levels of etheno adducts also are observed in colon polyps of patients with familial adenomatous polyposis38. In addition, the levels of εA and εC in white blood cell DNA are significantly higher in women consuming diets rich in polyunsaturated fatty acids compared with women consuming diets with high monounsaturated fatty acids39. Interestingly, comparable elevations are not detected in men consuming the same diets. Increased production of εA and εC is detected during the tumor promotion phase of the mouse skin two-stage model of carcinogenesis40. Chronic treatment with the phorbol ester tumor promoter, tetradecanoylphorbol acetate, leads to 12- and nine-fold increases of εA and εC, respectively. This increase in DNA damage correlates to induction of the fatty acid oxygenase, 8-lipoxygenase, by the phorbol ester40. Several exocyclic DNA adducts in human and animal tissue appear to arise by addition of α,β-unsaturated aldehydes to deoxyguanosine residues (Fig. 5). These adducts are all derivatives of 1,N2-propanodeoxyguanosine and form by addition of the exocyclic amino group of deoxyguanosine to the β-carbon of the unsaturated aldehyde followed by ring closure of the aldehyde carbon onto the imine nitrogen41,42. The aldehydes – acrolein, crotonaldehyde and 4-hydroxynonenal – arise by lipid peroxidation so the formation of these propano adducts is related to oxidative stress. Fourfold increases in the levels of the acrolein and crotonaldehyde adducts are detected in the oral tissue of smokers compared with non-smokers43. In rats, depletion of glutathione by administration of buthionine sulfoximine leads to two- and 15- to 21-fold increases in the levels of DNA adducts in liver derived from acrolein and crotonaldehyde, respectively44. Glutathione is an important scavenger of reactive electrophiles including α,β-unsaturated aldehydes, so its depletion increases their concentration. The pyrimidopurinone adduct, M1G, arises from the lipid oxidation product, malondialdehyde (Fig. 6; Ref. 23). This adduct also can be made by reaction of base propenals with DNA45. Base propenals are products of degradation of DNA by oxidizing agents such as the cancer chemotherapeutic agent, bleomycin. Quantitative studies indicate that base propenals are ~100-fold more reactive to DNA than malondialdehyde46. They are also highly mutagenic in the same frameshift tester strains of Salmonella typhimurium that are reverted by malondialdehyde46. Thus, the high levels of M1G that have been detected in certain tissues can arise not only from lipid peroxidation, but also from DNA degradation by endogenous agents that react analogously to bleomycin to form base propenals. It will be interesting to determine whether the agents that oxidize DNA to form M1G are the same oxidants that generate the poorly repaired 5′-cleaved AP sites detected in intact tissue by Nakamura et al.12

Review

216

TRENDS in Genetics Vol.17 No.4 April 2001

OH 3′

H2N

O

O O O P O O

5′

AP site AP endo

R R N O

3′

O O P O O

OH 3′

O OH O P O O O O P O O

O O O P O O

OH

5′

Detect Immunochemically

R = HN

H2O

O O P O O

5′

O O P O O

5′

dRP-lyase S

H N

OH

NH 3′

HN

O

O

ARP reagent

OH

O O O P O O

TRENDS in Genetics

Fig. 2. Repair and detection of AP sites. AP sites (blue) arise from spontaneous hydrolysis of purines and alkylated purines (e.g. 3-methyladenine and 7-methylguanine) and by glycosylase-catalyzed hydrolysis of damaged purines and pyrimidines (e.g. 8-oxo-G, 3-methyladenine, thymine glycol). AP sites are hydrolyzed by AP endonuclease then the deoxyribosyl-5′-phosphate group is removed by the dRP-lyase activity of DNA polymerase β. AP sites are detected by reaction of the hydroxylamine moiety of the ARP reagent (red) to form a stable oxime, which is detected immunochemically with biotin-specific reagents.

Damage at CpG sites

Considerable attention has focused on the cause of C→T transitions at CpG sites because this is a very common mutation, detected in a range of genetic diseases as well as in many cancers47,48. Numerous O

O

O H

H

H

H3C Acrolein O O

HO

Crotonaldehyde

Malondialdehyde

R C5H11

[OX] O2

OH O H

4-Hydroxynonenal [OX] R = glycerophospholipid C5H11

OH O O H

2,3-Epoxy-4-hydroxynonenal TRENDS in Genetics

Fig. 3. Aldehyde products of the oxidative degradation of polyunsaturated fatty acid residues in phospholipids. An arachidonic acid molecule is depicted for illustration. Many other products are generated in unsaturated fatty acid oxidation that are not depicted. The further conversion of 4-hydroxynonenal to its 2,3-epoxy derivative is also depicted. This epoxide is believed to be a source of the two carbons added to nucleoside bases to form etheno adducts.

http://tig.trends.com

hypotheses have been offered for the molecular events leading to this mutation, all of which emphasize the importance of methylation of cytosine residues. Methylation increases the rate of hydrolytic deamination and also increases the reactivity of neighboring guanines to electrophiles49. Although the rate of cytosine deamination in duplex DNA is extremely slow, hydrolysis of 5-methylcytosine is approximately twice as fast50. Genetic evidence suggests that the C→T transition in CpG sequences results from the enhanced hydrolysis of 5-methylcytosine to T combined with inefficient repair of T:G mismatches51. However, the possibility that oxidation of 5-methylcytosine contributes to the higher frequency of C→T transitions cannot be excluded. Oxygen radicals react with 5-methylcytosine to oxidize the 5,6-double bond; the intermediate product, 5-methylcytosine glycol, then deaminates to form thymine glycol (Fig. 7; Ref. 52). Thymine glycol and the related adducts, 5-hydroxy-uracil and uracil glycol, base-pair with A, so oxidation of 5-methylcytosine is expected to result in a C→T transition53. Base damage and chromosomal rearrangements

Rearrangement of large tracts of DNA can cause chromosomal translocations and losses of heterozygosity that are thought to be crucial for carcinogenesis54. Chromosomal alterations are initiated inter alia by double-strand breaks resulting from oxidative cleavage of the DNA backbone or enzymatic cleavage during chromatin remodeling (e.g. by topoisomerase II). DNA replication itself contributes about ten double-strand breaks per cell cycle in the form of stalled or blocked replication forks55. Double-strand breaks are specifically recognized by binding proteins, such as Ku autoantigen, hRad 52 and p53, which can stimulate double-strand break repair and halt cell-cycle progression to allow more time for repair to occur56,57. Because double-strand breaks are important endogenous premutagenic lesions, factors that lead to their formation, as well as aberrations in the pathways that repair them, add to the process of endogenous mutagenesis. AP sites and exocyclic adducts increase the rate of topoisomerase I- and topoisomerase II-catalyzed cleavage of DNA58,59. Enhanced cleavage by topoisomerase II is particularly interesting because it provides a direct route to the formation of doublestrand breaks. The stimulation of topoisomerase II activity is significantly greater with exocyclic adducts than with AP sites60,61. The lesions must be present within the topoisomerase cleavage site and enhanced cleavage results from an increased rate of cleavage by topoisomerase II rather than by slowed re-ligation62. Topoisomerase II consensus sequences are not stringent and many sites exist in the genome to enable rapid topological reorganization of chromatin during cell proliferation. This makes it likely that AP sites or

Review

TRENDS in Genetics Vol.17 No.4 April 2001

O

N N

N

εA→G

N

N

N 2,3-εG→A N

N

R

N

HN

εA→T

N R

N

O εC→T

N O

N

N

εC→A

N

N

H

R

1, N 2-εG→A

N

N

1, N 2-εG→T

R

TRENDS in Genetics

Fig. 4. Etheno adducts detected in human genomic DNA and the mutations that they induce.

exocyclic adducts can be generated in topoisomerase IIcleavable sequences, leading to the production of double-strand breaks. Defects in double-strand break repair genes, such as BRCA1 (breast cancer), BRCA2, NBS1 (Nijmegen breakage syndrome), ATM (ataxia telangiectasia – mutated) and DNA ligase IV have been implicated in increased cancer risk57,63. Endogenous DNA damage and genetic disease

Is there a relationship between the levels of endogenous DNA damage and increased risk of OH

O

O

O

N

N H N

N

N

Acrolein

HO

R

H

OH

N N

N

OH H

HO

4-Hydroxynonenal

H11C5

N H

N

N

N

H

N R

6-OH-8-Me-PdG

O N

N HO

C5H11

HO

R

8-OH-6-Me-PdG

O

R

N

+

H

Crotonaldehyde

N

CH3 O

O

N

H3C

H3C

N

6-OH-PdG

N

H

N H

8-OH-PdG

O

N

N

+

N

N R

8-OH-6-Hydroxyhexyl-PdG TRENDS in Genetics

Fig. 5. Propanodeoxyguanosine adducts formed by reaction of aldehydes with deoxyguanosine residues.

http://tig.trends.com

217

genetic disease? Extensive data are not available with which to address this question but the results of several studies are provocative. The exocyclic adduct N2,3-εG occurs endogenously in rodents, and its levels can be increased by treatment of animals with the environmental carcinogen vinyl chloride64. The background level of N2,3-εG in rat liver is nine adducts per 108 guanines; somewhat higher levels are present in human liver. When rats are exposed to 10 ppm vinyl chloride for 5 or 20 days, the levels of N2,3-εG increase 2.2- and 5.9-fold, respectively. Concentration-dependent increases in the steadystate levels of N2,3-εG also are observed in vinyl chloride-exposed rats. A statistically significant increase in the incidence of angiosarcomas (20% incidence at two years) occurs at an adduct level approximately tenfold higher than the level of N2,3-εG in untreated rat liver. Similar data relating levels of endogenous DNA damage to cancer risk are not available for human beings. However, recent studies of the levels of tamoxifen-derived DNA adducts in the endometrial tissue of women taking this drug for prevention of breast cancer provide critical dose-response information on the induction of DNA damage in a target tissue by a human carcinogen. In one study, adducts were detectable by [32P]-postlabeling in six of 13 women taking clinical doses of tamoxifen (20 mg per day; Ref. 65). No adducts were detected in the endometrial tissue of ten women not taking tamoxifen. The levels of the trans tamoxifen adducts in the six women in whom adducts were detected ranged from 0.5–8.3 per 108 bases whereas the levels of the cis adducts ranged from 0.4–4.8 per 108 bases. The risk of endometrial cancer in women taking this dose of tamoxifen increases fourfold over a five-year period relative to the risk in women not taking tamoxifen66. Thus, levels of tamoxifen adducts on the order of 1 per 107 bases are associated with an increased risk of developing endometrial cancer. Combined with the ability of tamoxifen to stimulate cell proliferation, DNA damage could be an important component of the carcinogenic activity of tamoxifen. Targeted deletions of genes coding for various glycosylases confirm the role of these proteins in repair of endogenous DNA damage and suggest that elevations in adduct levels can lead to increases in mutation67–71. Two groups have knocked out the Mmh/Ogg1 glycosylase gene responsible for removal of 8-oxo-G (Refs 67,68). Ogg1–/– mice appear normal and do not exhibit any dramatic phenotypes. The levels of 8-oxo-G in liver are higher in knockout mice relative to wild-type mice, although the magnitude of the increase is difficult to quantify accurately because of possible artifacts in the determinations. Klungland et al. estimate increased 8-oxo-G levels of 400 per genome (1.5 adducts per 107 basepair) in rapidly proliferating fibroblasts in culture and 15 000 per genome in nonproliferating livers from adult animals67. Elevated mutation frequencies at the lacI

Review

218

TRENDS in Genetics Vol.17 No.4 April 2001

O

O

O

H2N

R

N

HN

+

N

N

N

N N

N

N M1G

R = OH:malondialdehyde R = nucleobase:base propenal TRENDS in Genetics

Fig. 6. Pyrimidopurinone (M1G) adduct formed by reaction of deoxyguanosine residues with malondialdehyde or base propenals.

and gpt loci (introduced as transgenes) are apparent in liver67,68. The principal increase in mutations observed is in G→T transversions, which is consistent with a role for 8-oxo-G as the responsible lesion. Interestingly, no increase in lacI mutations is observed in testis67. The Aag/Mpg/Anpg gene, which is responsible for the repair of 3-methyladenine and several other endogenous DNA adducts (e.g. 7-methylguanine and εA) also has been knocked out69–71. As with the Ogg1 knockout, Aag–/– animals appear normal. The basal levels of 7-methylguanine in liver appear comparable in mutant and wild-type mice but, following treatment with methylnitrosourea, removal of 7-methylguanine is severely retarded in mutant mice69. Likewise, the frequencies of spontaneous mutations in the hprt gene measured in T lymphocytes are comparable in Aag–/– mice and wild-type animals, but following treatment with methylnitrosourea, the frequency of hprt mutations is elevated threefold in Aag–/– mice compared with wild-type mice69. The major mutations observed in Aag–/– mice are A→T transversions consistent with O H3C

NH N

NH2 H3C

O

H 2O Thymine

-NH3

N N

O [OX]

O

NH2 H3C HO HO

H2O

N

-NH3 N

O

H3C

NH

HO HO

N

O

Thymine glycol TRENDS in Genetics

Fig. 7. Hydrolytic or oxidative deamination of 5-methylcytosine residues to thymine or thymine glycol, respectively.

http://tig.trends.com

the involvement of 3-methyladenine as the premutagenic lesion. The studies with repair-deficient animals support the hypothesis that increased levels of endogenous adducts lead to increased frequencies of mutation, but they also illustrate the complexity associated with studying endogenous DNA damage and mutation. Examination of nuclear extracts from various tissues of Ogg1–/– animals establishes that this enzyme is the sole glycosylase acting on 8-oxo-G in vivo67,68. However, embryonic fibroblasts from knockout animals repair 8-oxo-G residues, albeit at a reduced rate, compared to wild-type animals. It is likely that transcription-coupled nucleotide-excision repair contributes to removal of 8-oxo-G from the transcribed strands of expressed genes and global nucleotide excision repair removes the remainder of the lesions72. The complementary relationship of these repair pathways provides multiple mechanisms for minimizing the steady-state adduct levels so that deletion of a single repair gene might not have as dramatic an effect on mutagenesis as anticipated. In addition, the low proliferative rate of many tissues could mask the mutagenic potential of DNA adducts that survive repair. With regard to human beings, increases in mutations in somatic cells are associated with some inflammatory conditions. For example, Hussain et al. recently detected higher frequencies of p53 mutations in inflamed colon tissue from patients with ulcerative colitis relative to uninvolved tissue from the same patients73. Increases in mutations might arise as a result of oxidative stress, as well as from increased cell division arising from compensatory replication due to cell death. Ulcerative colitis is a chronic inflammatory disease associated with an elevated risk of colon cancer. The occurrence of higher frequencies of mutations in inflamed tissue could represent a contributing factor to the increased risk. Similar observations have been reported for K-ras mutations in individuals with pancreatitis74 and for p53 mutations in individuals with Wilson’s disease or hemochromatosis75. Thus, it appears that endogenous oxidative stress can lead to an elevated frequency of mutations in target cells for carcinogenesis but not in non-target cells. Alternatively, the growth of mutant cells might be favored by the selective pressure provided by the complex milieu created by the inflammatory process, which includes cytokine release, altered gene expression, etc.76 Future directions Identification and measurement of adducts

It is likely that analytical methodology will continue to improve at a rapid pace, which will provide more sensitive and reliable ways to identify and quantitate endogenous DNA damage. The possibility that mass spectrometric techniques will be applied to the identification of previously unknown forms of DNA damage is particularly exciting. For example, DNA adducts that exhibit chromatographic properties

Review

TRENDS in Genetics Vol.17 No.4 April 2001

similar to bulky aromatic adducts have been detected in breast tissue77. The adducts are present in nonsmokers, so they seem not to be derived from tobacco constituents. Could they be stable adducts derived from quinones that are endogenous products of estrogen metabolism78,79? If these adducts are identified in human DNA samples, it might provide insight into potential causes of breast cancer. A recent report describes the ability of myeloperoxidase and eosinophil peroxidase to brominate cytosine and uracil to their 5-bromo derivatives. It will be interesting to determine whether these or other brominated bases are present as endogenous constituents of human DNA. If so, they could provide very useful biomarkers for inflammation-associated DNA damage80. Immunochemical assays have been developed for the quantification of individual DNA adducts that should be adaptable for high-throughput studies. For example, a robust immuno-slotblot method has been described for the detection of the pyrimidopurinone, M1G, that enables adduct quantitation to a limit of ~1 adduct per 108 nucleotides using 1 µg DNA81. Although immunochemical assays lack the absolute specificity of mass spectrometry-based methods, they are relatively inexpensive and are easily adapted to microtiter format. Thus, they are ideally suited for large population-based studies of adduct levels in a variety of DNA samples. Such studies can be designed to test novel hypotheses or to confirm and extend the results of earlier studies that employed more cumbersome analytical methods, which may have limited the number of subjects. For example, it would be interesting to confirm the association of high levels of certain endogenous exocyclic adducts in women consuming diets high in unsaturated fats39. Mechanisms of mutations Acknowledgements This article is dedicated to the memory of James and Elizabeth Miller.

Recently, several new DNA polymerases that appear to have important roles in lesion bypass have been discovered82. These polymerases are highly

References 1 Ames, B.N. and Saul, R.L. (1988) Cancer, aging, and oxidative DNA damage. In Theories of Carcinogenesis (Iverson, O.H., ed.), pp. 203–220, Hemisphere Publishing 2 Marnett, L.J. (2000) Oxyradicals and DNA damage. Carcinogenesis 21, 361–370 3 Burcham, P.C. (1999) Internal hazards: baseline DNA damage by endogenous products of normal metabolism. Mutat. Res. 443, 11–36 4 Farmer, P.B. (1999) Studies using specific biomarkers for human exposure assessment to exogenous and endogenous chemical agents. Mutat. Res. 428, 69–81 5 Randerath, K. et al. (1999) Bulky endogenous DNA modifications (I-compounds) – possible structural origins and functional implications. Mutat. Res. 424, 183–194 6 Gupta, R.C. and Lutz, W.K. (1999) Background DNA damage for endogenous and unavoidable exogenous carcinogens: a basis for spontaneous cancer incidence? Mutat. Res. 424, 1–8 http://tig.trends.com

219

distributive; they only insert one or a few bases at the site of lesions, then they dissociate and are replaced by replicative polymerases83,84. These novel DNA polymerases exhibit very low fidelity, which makes it likely that they are responsible for DNA damageinduced mutations85,86. In fact, their low fidelity suggests their expression and catalytic activity must be tightly regulated. Consequences of DNA damage

We need to accumulate much more information on the biological consequences of DNA damage. The main focus of experiments conducted to date has been on the ability of adducts to induce basepair substitutions and frameshift mutations in sitespecific mutagenesis assays. However, DNA damage is linked to a much broader range of cellular responses, and the experimental tools to evaluate the role of defined forms of damage in triggering these responses are just becoming available. For example, Pandya et al. recently described an assay to determine the recombinogenic properties of single DNA adducts carried on plasmids87. On a more global level, soon it will be possible to test the role of DNA adducts in triggering cellular changes in gene expression and protein modification. Microarray and proteomic techniques can be used to determine the quantitative and qualitative changes in cellular macromolecules induced by DNAdamaging agents. It is frequently assumed that all changes in gene expression, cell-cycle control and apoptosis induced by free radicals and electrophiles result from DNA damage. In fact, DNA is a much less important target for reactive compounds than protein and RNA, so it is likely that some of the phenotypic changes induced by chemical treatment of cells do not result from DNA damage. The availability of cells bearing targeted deletions in individual DNA repair genes provides a powerful tool for functional genomic approaches to evaluating the cellular effects of DNA damage88.

7 Memisoglu, A. and Samson, L. (2000) Baseexcision repair in yeast and mammals. Mutat. Res. 451, 39–51 8 Kubo, K. et al. (1992) A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion. Biochemistry 31, 3703–3708 9 Nakamura, J. et al. (1998) Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions. Cancer Res. 58, 222–225 10 Lindahl, T. (1993) Instability and decay of the primary structure of DNA. Nature 362, 709–715 11 Nakamura, J. and Swenberg, J.A. (1999) Endogenous apurinic/apyrimidinic sites in genomic DNA of mammalian tissues. Cancer Res. 59, 2522–2526 12 Nakamura, J. et al. (2000) 5′-Nicked apurinic/apyrimidinic sites are resistant to betaelimination by beta-polymerase and are persistent in human cultured cells after oxidative

stress. J. Biol. Chem. 275, 5323–5328 13 Gentil, A. et al. (1990) Mutagenic properties of a unique abasic site in mammalian cells. Biochem. Biophys. Res. Commun. 173, 704–710 14 Lawrence, C.W. et al. (1990) Mutation frequency and spectrum resulting from a single abasic site in a single-stranded vector. Nucleic Acids Res. 18, 2153–2157 15 Jackson, A.L. et al. (1998) Induction of microsatellite instability by oxidative DNA damage. Proc. Natl. Acad. Sci. U. S. A. 95, 12468–12473 16 Atamna, H. et al. (2000) A method for detecting abasic sites in living cells: age-dependent changes in base-excision repair. Proc. Natl. Acad. Sci. U. S. A. 97, 686–691 17 Wang, D. et al. (1998) Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat. Res. Fundam. Mol. Mech. Mutagen. 400, 99–115 18 Demple, B. and Harrison, L. (1994) Repair of oxidative damage to DNA. Annu. Rev. Biochem. 63, 915–948

220

Review

19 Le Page, F. et al. (1999) Repair and mutagenesis survey of 8-hydroxyguanine in bacteria and human cells. Biochimie 81, 147–153 20 Beckman, K.B. and Ames, B.N. (1999) Endogenous oxidative damage of mtDNA. Mutat. Res. 424, 51–58 21 Cadet, J. et al. (1998) Facts and artifacts in the measurement of oxidative base damage to DNA. Free Radic. Res. 29, 541–550 22 Senturker, S. and Dizdaroglu, M. (1999) The effect of experimental conditions on the levels of oxidatively modified bases in DNA as measured by gas chromatography-mass spectrometry: how many modified bases are involved? Prepurification or not? Free Radic. Biol. Med. 27, 370–380 23 Marnett, L.J. (1999) Lipid peroxidation-DNA damage by malondialdehyde. Mutat. Res. 424, 83–95 24 Singer, B. and Bartsch, H. (1999) Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis, International Agency for Research on Cancer 25 Fink, S.P. et al. (1997) Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde. Proc. Natl. Acad. Sci. U. S. A. 94, 8652–8657 26 Moriya, M. et al. (1994) Mutagenic potency of exocyclic DNA adducts: marked differences between Escherichia coli and simian kidney cells. Proc. Natl. Acad. Sci. U. S. A. 91, 11899–11903 27 Pandya, G.A. and Moriya, M. (1996) 1,N6ethenodeoxyadenosine, a DNA adduct highly mutagenic in mammalian cells. Biochemistry 35, 11487–11492 28 Basu, A.K. et al. (1993) Mutagenic and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-ethenoadenine, 3,N4-ethenocytosine, and 4-amino-5-(imidazol-2-yl)imidazole. Biochemistry 32, 12793–12801 29 Mao, H. et al. (1999) Duplex DNA catalyzes the chemical rearrangement of a malondialdehyde deoxyguanosine adduct. Proc. Natl. Acad. Sci. U. S. A. 96, 6615–6620 30 Vanderveen, L.A. et al. Evaluation of the mutagenic potential of the principal DNA adduct of acrolein. J. Biol. Chem. (in press) 31 Yang, I-Y. et al. Responses to the major acroleinderived deoxyguanosine adduct in Escherichia coli. J. Biol. Chem. (in press) 32 de los Santos, C. et al. NMR characterization of a DNA duplex containing g-(OH)-1,N2propanodeoxyguanosine: the major acroleinderived deoxyguanosine adduct. J. Biol. Chem. (in press) 33 Nair, J. et al. (1999) Etheno DNA-base adducts from endogenous reactive species. Mutat. Res. 424, 59–69 34 Ham, A.J. et al. (1999) Immunoaffinity/gas chromatography/high-resolution mass spectrometry method for the detection of N(2),3ethenoguanine. Chem. Res. Toxicol. 12, 1240–1246 35 Chung, F.L. et al. (1996) Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts. Carcinogenesis 17, 2105–2111 36 Sodum, R.S. and Chung, F-L. (1988) 1,N2ethenodeoxyguanosine as a potential marker for DNA adduct formation by trans-4-hydroxy-2nonenal. Cancer Res. 48, 320–323 37 Nair, J. et al. (1998) Lipid peroxidation-induced etheno-DNA adducts in the liver of patients with the genetic metal storage disorders http://tig.trends.com

TRENDS in Genetics Vol.17 No.4 April 2001

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54 55

56

Wilson’s disease and primary hemochromatosis. Cancer Epidemiol. Biomarkers Prev. 7, 435–440 Schmid, K. et al. (2000) Increased levels of promutagenic etheno-DNA adducts in colonic polyps of FAP patients. Int. J. Cancer 87, 1–4 Nair, J. et al. (1997) High dietary ω-6 polyunsaturated fatty acids drastically increase the formation of etheno-DNA base adducts in white blood cells of female subjects. Cancer Epidemiol. Biomarkers Prev. 6, 597–601 Nair, J. et al. (2000) Promutagenic etheno-DNA adducts in multistage mouse skin carcinogenesis: correlation with lipoxygenase-catalyzed arachidonic acid metabolism. Chem. Res. Toxicol. 13, 703–709 Chung, F.L. et al. (1999) Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts. Mutat. Res. 424, 71–81 Chung, F.L. et al. (2000) Deoxyguanosine adducts of t-4-hydroxy-2-nonenal are endogenous DNA lesions in rodents and humans: detection and potential sources. Cancer Res. 60, 1507–1511 Nath, R.G. et al. (1998) 1,N2propanodeoxyguanosine adducts: potential new biomarkers of smoking-induced DNA damage in human oral tissue. Cancer Res. 58, 581–584 Nath, R.G. et al. (1997) Effect of glutathione depletion on exocyclic adduct levels in the liver DNA of F344 rats. Chem. Res. Toxicol. 10, 1250–1253 Dedon, P.C. et al. (1998) Indirect mutagenesis by oxidative DNA damage: formation of the pyrimidopurinone adduct of deoxyguanosine by base propenal. Proc. Natl. Acad. Sci. U. S. A. 95, 11113–11116 Plastaras, J.P. et al. (2000) Reactivity and mutagenicity of endogenous DNA oxopropenylating agents: base propenals, malondialdehyde, and Nε-oxopropenylysine. Chem. Res. Toxicol. 13, 1235–1242 Cooper, D.N. and Youssoufian, H. (1988) The CpG dinucleotide and human genetic disease. Hum. Genet. 78, 151–155 Hainaut, P. and Hollstein, M. (2000) p53 and human cancer: the first ten thousand mutations. Adv. Cancer Res. 77, 81–137 Pfeifer, G.P. (2000) p53 mutational spectra and the role of methylated CpG sequences. Mutat. Res. 450, 155–166 Shen, J.C. et al. (1994) The rate of hydrolytic deamination of 5-methylcytosine in doublestranded DNA. Nucleic Acids Res. 22, 972–976 Lutsenko, E. and Bhagwat, A.S. (1999) Principal causes of hot spots for cytosine to thymine mutations at sites of cytosine methylation in growing cells. A model, its experimental support and implications. Mutat. Res. 437, 11–20 Zuo, S. et al. (1995) Oxidative damage to 5methylcytosine in DNA. Nucleic Acids Res. 23, 3239–3243 Kreutzer, D.A. and Essigmann, J.M. (1998) Oxidized, deaminated cytosines are a source of C→T transitions in vivo. Proc. Natl. Acad. Sci. U. S. A. 95, 3578–3582 Lengauer, C. et al. (1998) Genetic instabilities in human cancers. Nature 396, 643–649 Haber, J.E. (1999) DNA recombination: the replication connection. Trends Biochem. Sci. 24, 271–275 Haber, J.E. (2000) Partners and pathways repairing a double-strand break. Trends Genet. 16, 259–264

57 Karran, P. (2000) DNA double strand break repair in mammalian cells. Curr. Opin. Genet. Dev. 10, 144–150 58 Kingma, P.S. and Osheroff, N. (1998) The response of eukaryotic topoisomerases to DNA damage. Biochim. Biophys. Acta 1400, 223–232 59 Pourquier, P. et al. (1997) Effects of uracil incorporation, DNA mismatches, and abasic sites on cleavage and religation activities of mammalian topoisomerase I. J. Biol. Chem. 272, 7792–7796 60 Sabourin, M. and Osheroff, N. (2000) Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions. Nucleic Acids Res. 28, 1947–1954 61 Kingma, P.S. et al. (1997) Spontaneous DNA lesions poison human topoisomerase IIalpha and stimulate cleavage proximal to leukemic 11q23 chromosomal breakpoints. Biochemistry 36, 5934–5939 62 Kingma, P.S. and Osheroff, N. (1997) Spontaneous DNA damage stimulates topoisomerase II-mediated DNA cleavage. J. Biol. Chem. 272, 7488–7493 63 Riballo, E. et al. (1999) Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient. Curr. Biol. 9, 699–702 64 Swenberg, J.A. et al. (1999) Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesis. In Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis (Singar, B. and Bartsch, H., eds), pp. 29–43, International Agency for Research on Cancer 65 Shibutani, S. et al. (1999) Tamoxifen-DNA adducts detected in the endometrium of women treated with tamoxifen. Chem. Res. Toxicol. 12, 646–653 66 Bernstein, L. et al. (1999) Tamoxifen therapy for breast cancer and endometrial cancer risk. J. Natl. Cancer Inst. 91, 1654–1662 67 Klungland, A. et al. (1999) Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage. Proc. Natl. Acad. Sci. U. S. A. 96, 13300–13305 68 Minowa, O. et al. (2000) Mmh/Ogg1 gene inactivation results in accumulation of 8-hydroxyguanine in mice. Proc. Natl. Acad. Sci. U. S. A. 97, 4156–4161 69 Elder, R.H. et al. (1998) Alkylpurine-DNA-Nglycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate. Mol. Cell. Biol. 18, 5828–5837 70 Engelward, B.P. et al. (1997) Base-excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase. Proc. Natl. Acad. Sci. U. S. A. 94, 13087–13092 71 Hang, B. et al. (1997) Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine. Proc. Natl. Acad. Sci. U. S. A. 94, 12869–12874 72 Le Page, F. et al. (2000) Transcription coupled repair of 8-oxoguanine in murine cells: the ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences. Proc. Natl. Acad. Sci. U. S. A. 97, 8397–8402 73 Hussain, S.P. et al. (2000) Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease. Cancer Res. 60, 3333–3337 74 Brentnall, T.A. et al. (1995) Microsatellite instability and K-ras mutations associated with pancreatic adenocarcinoma and pancreatitis. Cancer Res. 55, 4264–4267

Review

75 Hussain, S.P. et al. (2000) Increased p53 mutation load in nontumorous human liver of Wilson disease and hemochromatosis: Oxyradical overload diseases. Proc. Natl. Acad. Sci. U. S. A. 97, 12770–12775 76 Ambs, S. et al. (1998) p53 and vascular endothelial growth factor regulate tumor growth of NOS2-expressing human carcinoma cells. Nat. Med. 4, 1371–1376 77 Li, D. et al. (1999) DNA adducts in normal tissue adjacent to breast cancer: a review. Cancer Detect. Prev. 23, 454–462 78 Liehr, J.G. (2000) Is estradiol a genotoxic mutagenic carcinogen? Endocrinol. Rev. 21, 40–54 79 Cavalieri, E. et al. (2000) Estrogens as endogenous genotoxic agents – DNA adducts and mutations. J. Natl. Cancer Inst. Monogr. 75–93

TRENDS in Genetics Vol.17 No.4 April 2001

80 Henderson, J.P. et al. Production of brominating intermediates by myeloperoxidase: a transhalogenation pathway for generating mutagenic nucleobases during inflammation. J. Biol. Chem. (in press) 81 Leuratti, C. et al. (1998) Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay. Carcinogenesis 19, 1919–1924 82 Woodgate, R. (1999) A plethora of lesionreplicating DNA polymerases. Genes Dev. 13, 2191–2195 83 Tang, M. et al. (1999) UmuD′(2)C is an errorprone DNA polymerase, Escherichia coli pol V. Proc. Natl. Acad. Sci. U. S. A. 96, 8919–8924 84 Maor-Shoshani, A. et al. (2000) Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basis for SOS

221

85

86

87

88

89

untargeted mutagenesis. Proc. Natl. Acad. Sci. U. S. A. 97, 565–570 Matsuda, T. et al. (2000) Low fidelity DNA synthesis by human DNA polymerase-eta. Nature 404, 1011–1013 Johnson, R.E. et al. (2000) Fidelity of human DNA polymerase eta. J. Biol. Chem. 275, 7447–7450 Pandya, G.A. et al. (2000) Escherichia coli responses to a single DNA adduct. J. Bacteriol. 182, 6598–6604 Jelinsky, S.A. et al. (2000) Regulatory networks revealed by transcriptional profiling of damaged Saccharomyces cerevisiae cells: rpn4 links baseexcision repair with proteasomes. Mol. Cell. Biol. 20, 8157–8167 Drake, J.W. et al. (1998) Rates of spontaneous mutation. Genetics 148, 1667–1686

The myotubularin family: from genetic disease to phosphoinositide metabolism Jocelyn Laporte, François Blondeau, Anna Buj-Bello and Jean-Louis Mandel The myotubularin-related genes define a large family of eukaryotic proteins, most of them initially characterized by the presence of a ten-amino acid consensus sequence related to the active sites of tyrosine phosphatases, dualspecificity protein phosphatases and the lipid phosphatase PTEN. Myotubularin (hMTM1), the founder member, is mutated in myotubular myopathy, and a close homolog (hMTMR2) was recently found mutated in a recessive form of Charcot–Marie–Tooth neuropathy. Although myotubularin was thought to be a dual-specificity protein phosphatase, recent results indicate that it is primarily a lipid phosphatase, acting on phosphatidylinositol 3-monophosphate, and might be involved in the regulation of phosphatidylinositol 3-kinase (PI 3-kinase) pathway and membrane trafficking. Myotubularin proteins and human disease

J. Laporte F. Blondeau A. Buj-Bello J-L. Mandel* Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP163, 67404 Illkirch Cedex, C.U. de Strasbourg, France. *e-mail: mtm@ titus.u-strasbg.fr

The myotubularin phosphatase family comprises at least ten human members, six in Drosophila melanogaster and Caenorhabditis elegans, and one in yeast. Myotubularin, the founder member of this novel family, is a 603 amino acid long protein encoded by the human hMTM1 gene. It was identified by positional cloning as being mutated in X-linked myotubular myopathy (XLMTM, see Glossary), a very severe congenital myopathy1. This disease, first described in 1969, affects about one in 50 000 new-born males. Patients have a severe hypotonia at birth, and most of them die from hypoventilation (respiratory failure) within the first months of life2. However, a subset of patients survive for several years3–5, some even showing improvement of their clinical condition. Rare patients with milder forms are still alive past the age of 40 years.

The disease is characterized by the presence of disorganized skeletal muscle fibers that contain centrally located nuclei, resembling myotubes (Fig. 1). These nuclei are surrounded by mitochondria and other organelles. This pattern, which constitutes the key diagnostic feature, suggests that the disease is caused by a defect either in late muscle maturation (a step involving migration of nuclei to the periphery) or in the structural organization of the fibers6. Patients’ myoblasts undergo apparently normal fusion into myotubes in culture7, and these myotubes contract and differentiate as normal muscle cells when innervated (O. Dorchies, unpublished). Occurrence in a few patients of an otherwise very rare hemorrhagic manifestation in liver (peliosis), suggests that extramuscular tissues can also be affected5. More than 130 different hMTM1 mutations have been found in patients from 198 families8. These include 37 missense mutations, almost all of which affect residues conserved in a Drosophila ortholog that shares 54% amino acid identity with the human protein (Fig. 2). Three recurrent mutations affect 17% of patients, and more than 80% of the mutations (including some missense mutations) lead to a loss of myotubularin9. The high mutation heterogeneity is expected for such a severe X-linked disease. Affected males cannot reproduce, therefore about a third of mutated alleles are lost per generation and are replaced by a similar proportion of new mutations,

http://tig.trends.com 0168–9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02245-4