Nuclease P1 Digestion Combined with Tandem Mass Spectrometry for

One of the best approaches to this problem involves the study of DNA containing site-specific, chemically pure modifications (1). Although .... to obt...
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Chem. Res. Toxicol. 1999, 12, 1077-1082

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Nuclease P1 Digestion Combined with Tandem Mass Spectrometry for the Structure Determination of DNA Photoproducts Yinsheng Wang, John-Stephen Taylor,* and Michael L. Gross* Department of Chemistry, Washington University, St. Louis, Missouri 63130 Received May 14, 1999

UV irradiation of oligodeoxynucleotides at 254 nm generates several different types of DNA photoproducts, such as cis-syn cyclobutane pyrimidine dimers, pyrimidine[6-4]pyrimidone photoproducts and their Dewar valence isomers, and thymine-adenine photoproducts (TA*). Nuclease P1 degrades the oligodeoxynucleotide photoproducts to small photoproduct-containing trinucleotides which are more amenable to tandem mass spectrometry (MS/MS) and HPLC. Product-ion mass spectra of these digestion products give characteristic fragmentations, allowing us to identify quickly the types of photomodifications. The results also show that mass spectrometry will be a tool for studying enzyme reaction mechanisms because it can determine rapidly and with high sensitivity the structures of the products that are generated.

Introduction Human DNA constantly undergoes deleterious modifications, either spontaneously or by the action of physical or chemical agents, some of which can lead to cancer. One goal in cancer research is to determine the structures of those modifications that lead to cancer and to elucidate the mechanisms by which these modifications do so. One of the best approaches to this problem involves the study of DNA containing site-specific, chemically pure modifications (1). Although methods have been developed for constructing DNA substrates containing site-specific modifications, it is not always easy to confirm the identity and purity of the modified DNA being used for the biological structure-activity studies, especially for longer DNA molecules. Another limitation is that most of the modifications being studied correspond to the most frequently produced modification, and little is known about the structures of less frequently produced modifications that may be equally if not more carcinogenic. Our goal is to develop a general method for the structure determination of DNA modifications. The method should be useful for assaying the identity and purity of modified DNA used in structure-activity studies and for discovering and determining the structure of new modifications of DNA. We are particularly interested in DNA photoproducts because they are linked to skin cancer, which is the most prevalent form of cancer (2-4). DNA photoproducts, such as the cis-syn cyclobutane pyrimidine dimer ([c,s]), the pyrimidine[6-4]pyrimidone product ([6-4]) (5) and its Dewar valence isomer, and the thymine-adenine photoproduct (TA*) (6, 7), are produced upon UV irradiation (structures of these photoproducts are shown in Figure 1). These modifications have been implicated in cell lethality, mutagenesis, and carcinogenesis (8-10). * To whom correspondence should be addressed: Department of Chemistry, Campus Box 1134, Washington University, St. Louis, MO 63130. Fax: (314) 935-7484. E-mail: [email protected].

Figure 1. Structures of the base portions of DNA photoproducts formed at TT and TA sites.

One of the most sensitive techniques for quantifying DNA photoproduct formation in vitro or in vivo is the 32 P postlabeling assay (11-17), a highly sensitive technique that is based on enzymatically degrading the modified DNA, radiolabeling the digestion products, and analyzing by two-dimensional thin-layer chromatography or by HPLC. Although the method is very sensitive, it does not provide structural information in the absence of suitable reference compounds. One-dimensional 1H NMR can be used to determine the structure of a photoproduct, but more than 10 nmol of sample is usually needed (18). The conventional formic acid hydrolysis assay (19) for the quantitation of DNA photoproducts provides no structural information, and it is not suitable for photomodifications that are acid labile or that are unknown. Mass spectrometry (MS)1 is a sensitive technique, and tandem mass spectrometry (MS/MS) can provide struc-

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In this article, we describe the use of nuclease P1 digestion followed by MS/MS analysis in identifying quickly the types of photomodifications in ODNs. Because mass spectrometry is highly sensitive and can provide structure information, we expect that it can be used to help elucidate the mechanism of substrate recognition and interactions by a variety of DNA processing enzymes. To this end, we compare the results of our study on the degradation of photodamaged ODNs by nuclease P1 with those obtained by X-ray crystallography (30, 31) and other biochemical studies (25-27, 32, 33) and show that the products that are formed are consistent with the mechanism proposed in a recent X-ray study (30).

Experimental Procedures Figure 2. HPLC traces for the separation of a UVC photolysis mixture of d(GTATTAT).

ture information. Our first approach to characterizing photodamaged DNA was to use MS/MS to examine ions from photoproduct-containing oligodeoxynucleotides (ODNs) that had been separated by HPLC and introduced to the spectrometer by fast atom bombardment (FAB) (18) or electrospray ionization (ESI) (20). The HPLC separation of the isomeric photoproducts, however, becomes very difficult or impossible when the DNA sequences are too long. In addition, the sensitivity of MS/ MS decreases and the complexity of fragmentation increases as molecular size increases. A significant improvement can be achieved if the modified ODNs can be degraded to smaller ones that contain the photoproducts. Those small ODNs should be more readily separable by HPLC and be more amenable to MS/MS than the larger, parental ones. An early example of this approach, but without MS/MS, is by Linscheid and co-workers (21), who used the unspecific nuclease, benzon nuclease, to digest DNA samples to small ODNs (2-8-mer) for capillary zone electrophoresis (CZE)/mass spectrometry analysis. The enzyme nuclease P1 from Penicillium citrinum, which has combined endo- and exonuclease activities and hydrolyzes single-stranded nucleic acids to 5′-mononucleotides (22), has often been used in conjunction with other enzymes to degrade DNA in postlabeling and HPLC assays of damaged DNA (23). Nuclease P1 digestion followed by MS/MS was recently used in the characterization of biotin-labeled ODNs (24). When biotin was attached to the middle or either end of the ODNs, different digestion products were produced. In another example, the synthetic oxazolone (25)-, 4-hydroxy-8-oxo4,8-dihydro-2′-deoxyguanosine (26)-, and (5′S)-cycloAdocontaining ODNs (27) were digested by nuclease P1 followed by alkaline phosphatase to give lesion-containing mono-, di-, and trinucleotides, respectively, and their structures were assigned on the basis of the molecular weight determination. The combination of DNase I followed by nuclease P1 digestions and CZE-ESI/MS/MS has also been used to characterize the adducts formed by phenyl glycidyl ether (28, 29); modifications on both the nucleic acid base and phosphate were identified. 1 Abbreviations: ODN, oligodeoxynucleotide; TEAA, triethylammonium acetate; CZE, capillary zone electrophoresis; MS, mass spectrometry; MS/MS, tandem mass spectrometry; FAB, fast atom bombardment; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization; PSD, post-source decay; UVC, ultraviolet radiation from 240 to 280 nm.

Oligodeoxynucleotides d(GTATTAT) and d(GGCTATAA) were obtained from Integrated DNA Technologies, Inc. (Coralville, IA), and trinucleotides d(pTAT) and d(pTTA) were obtained from the Nucleic Acids Chemistry Laboratory of Washington University. The preparation of the oligonucleotide photoproducts was described previously (20). Briefly, an oligodeoxynucleotide (ODN) was dissolved in H2O and degassed with N2. The solution was dispersed in a Petri dish, moved to a N2-filled zip-lock bag, irradiated on ice for 2 h, and dried with a Speed-Vacuum. The HPLC separation was carried out on a 4.6 mm i.d. Dynamax C18 reverse-phase column (Varian, Walnut Creek, CA). The flow rate was set at 0.5 mL/min, and a 40 min gradient of 6 to 12% CH3CN in 100 mM triethylammonium acetate (TEAA) was used. TEAA was used instead of a phosphate buffer so that no desalting step was needed to prepare the sample for mass spectrometric analysis. The separation traces of the UVC photolysis products of d(GTATTAT) (Figure 2) showed that the four major photoproducts, d(GTATT[ ]AT), d(GT[ ]ATTAT), d(GTAT[c,s]TAT), and d(GTAT[6-4]TAT), can be cleanly separated from each other as well as from the starting material. The identities of the photoproducts were confirmed by NMR, UV, and the acid hydrolysis fluorescence assay (18). The matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) experiments were carried out on a Voyager DERP instrument (Perseptive Biosystems, Framingham, MA). The matrix was a saturated solution of R-cyano-4-hydroxycinnamic acid in 2/1 (v/v) acetonitrile/H2O. The spectra from approximately 20 and 80 laser shots were averaged to obtain MALDI and MALDI post-source decay (PSD) spectra, respectively. ESI experiments were carried out on an LCQ ion trap mass spectrometer (Finnigan, San Jose, CA). The instrument conditions for MALDI and ESI were described previously (20). Nuclease P1 was procured from Boehringer-Mannheim (Mannheim, Germany) and used without further purification. For all the digestion reactions, no additional buffer was used except that used during the production of the enzyme. A stock solution of nuclease P1 was prepared by dissolving 1 mg of enzyme (about 300 units) in 300 µL of double-distilled water (ddH2O). For digestion, a 10-fold dilution of the stock solution (0.1 unit/µL) in ddH2O was used. For MALDI analysis, the digestions were carried out directly on the MALDI plate by mixing 0.5 µL of 10 pmol/µL oligodeoxynucleotide with 0.5 µL of 0.1 unit/µL nuclease P1. After incubation for 5 min, the matrix solution was applied to the reaction mixture, and the sample was introduced into the mass spectrometer. For ESI analysis, the digestions were carried out at room temperature by applying 5 µL of 0.1 unit/ µL nuclease P1 to 5 µL of 10 pmol/µL oligonucleotide. After incubation for 5 min, the samples were introduced into the mass spectrometer. For HPLC analysis of photolysis mixtures, 5 units of nuclease P1 was applied to ∼10 nmol of the photolysis mixtures, and the digestion was carried out overnight at 37 °C.

Results and Discussion We first tested the enzyme behavior of nuclease P1 with the authentic photodamaged oligodeoxynucleotides

P1 Digestion and MS/MS for DNA Photoproducts

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Figure 3. Negative ion MALDI-TOF mass spectra of the nuclease P1 digestion products of d(GTAT[c,s]TAT) (a) and d(GGCTAT[ ]AA) (b) and negative ion ESI mass spectrum of the nuclease P1 digestion product of d(GTAT[6-4]TAT) (c).

Figure 4. Product-ion spectra (MS/MS) (negative ion) of nuclease P1 digestion products of photomodified DNA such as (a) d(GT[ ]ATTAT), (b) d(GTATT[ ]AT), and (d) d(GGCTAT[ ]AA) and the MS/MS spectrum of d(pTAT) (c). The term “- H” is omitted from the ion designations to save space.

(ODNs). The six photomodified ODNs, d(GT[ ]ATTAT), d(GTATT[ ]AT), d(GTAT[c,s]TAT), d(GTAT[6-4]TAT), d(GGCT[ ]ATAA), and d(GTAT[Dewar]TAT), after digestion with nuclease P1, gave a common product, seen at m/z 938, whereas the digestion of d(GGCTAT[ ]AA) gave a product seen at m/z 947 in the negative ion MALDI or ESI spectra. Figure 3 shows the negative ion MALDI spectra for nuclease P1 digestion products of d(GTAT[c,s]TAT) and d(GGCTAT[ ]AA) and the negative ion ESI spectrum of the nuclease P1 digestion product of d(GTAT[6-4]TAT) as examples. In the ESI spectrum, the [M + Na - 2H]- and [M + 2Na - 3H]- ions were also observed in addition to the [M - H]- ion of the digestion product. Adduction with sodium ions occurs probably because there are salts in the enzyme. The molecular weights of those digestion products, especially those of d(GT[ ]ATTAT) and d(GGCT[ ]ATAA), are consistent with their identities as 5′-phosphorylated trinucleotides with a normal base 3′ to the modification site. Product-Ion Spectra of Digestion Products. To confirm further the identities of the nuclease P1 digestion products of d(GT[ ]ATTAT) and d(GTATT[ ]AT), we used ESI to obtain [M - H]- ions and studied them with MS/ MS. The digestion products of d(GT[ ]ATTAT) and d(GTATT[ ]AT) gave nearly the same fragment ions in the product-ion spectra (panels a and b of Figure 4). The most distinctive feature of the two spectra is that the most abundant fragment ion is [pT[ ]pA - H - H2O]- at m/z 616. For comparison, we obtained an authentic

sample of d(pTAT) and submitted it to MS/MS. The product-ion spectrum of undamaged d(pTAT) (Figure 4c) exhibits more fragment ions than those of the digestion products of two TA* photoproducts. The formation of the TA* photoproduct causes the formation of [pT[ ]pA - H2O - H]- to be much more favorable than the formation of other ions. The linkage between the two bases also prevents the formation of the [pT[ ]pA - H - Ade H2O]- ion (where Ade is adenine), whereas this ion is abundant in the product-ion spectrum of d(pTAT). Comparison of the product-ion spectrum of the undamaged trinucleotide and those of the digestion products of photodamaged ODNs confirms the identities of the digestion products. The fragmentation of the nuclease P1 digestion product of d(GGCTAT[ ]AA) also gives a most abundant fragment ion at m/z 616 (Figure 4d), which supports our assignment of this ion as [pT[ ]pA - H2O H]-. We now turn to the product-ion spectra for the ESIproduced [M - H]- ion of the digestion products of the three photoproducts formed at adjacent thymine sites in d(GTATTAT). These spectra are different from those of the TA* photoproducts. The major fragment ions of the nuclease P1 digestion product of the [c,s] isomer are [M - H - H2O]- at m/z 920, [M - H - Ade]- at m/z 803, [M - H - Ade - H2O]- at m/z 785, [M - H - Ade - H2O HPO3]- at m/z 705, [M - H - Ade - 2H2O - HPO3]- at m/z 687, and [pT[c,s]pT - H - H2O]- at m/z 607 (Figure 5a). In addition to these fragment ions, the product-ion

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spectrum of the digestion product of the [6-4] isomer exhibits an additional and highly abundant fragment ion at m/z 825 (denoted with an asterisk in Figure 5b). The loss of 113 mass units also occurred upon high-energy

Figure 5. Product-ion spectra (MS/MS) of nuclease P1 digestion products of photomodified DNA such as (a) d(GTAT[c,s]TAT) and (b) d(GTAT[6-4]TAT) and the MS/MS spectrum of d(pTTA) (c).

Figure 6. HPLC trace for the separation of nuclease P1 digestion products of the photolysis mixture of d(GTATTAT). Buffer A was 100 mM triethylammonium acetate in water and buffer B 30% acetonitrile in buffer A. The gradient lasted for 40 min and was from 0 to 25% B in A. A 250 mm × 4.6 mm Dynamax C-18 reverse phase column was used. A 260 nm UV detector was used for detection.

collisional activation of the FAB-produced [M + H]+ ion of d(T[6-4]T) (34), and the loss was thought to be part of the pyrimidine moiety accompanied by hydrogen rearrangement (8). The neutral is probably C4H3O3N, and a mechanism for the formation of the ion is shown in Scheme 1. The product-ion spectrum of the digestion product of the Dewar isomer is quite similar to that of the [6-4] isomer except that the abundance of the fragment ion at m/z 825 of the Dewar isomer is about 25-30% of that of the [6-4] isomer (data not shown). By way of comparison, the product-ion spectrum of the [M - H]- ion of undamaged d(pTTA) (Figure 5c) shows the formation of the [M - Thy - H2O - HPO3 - H]- ion (where Thy is thymine). The linkage in the TT dimer once again prevents the formation of this ion, which further confirms that the digestion products are photoproductcontaining trinucleotides. MALDI-PSD gave similar results (data not shown). Because the HPLC separation of the DNA photoproducts becomes difficult or impossible when the DNA strand is long, P1 digestion should give products that are more readily separated. The photoproduct-containing trinucleotides can be separated from each other and from the mononucleotides, as shown in the HPLC traces of the photolysis mixture of d(GTATTAT) after digestion (Figure 6). The HPLC eluents were collected and submitted to ESI-MS and MS/MS analyses, which reveal that the eluents are d(pT) at a retention time of 22.7 min, dG at 29.3 min, d(pA) at 32.9 min, d(pT[ ]pApT) at 38.6 min, d(pT[c, s]pTpA) at 43.4 min, and d(pTp[6-4]TpA) at 43.9 min. It should be noted that the unmodified base that is located 3′ to the photodamaged site in the trinucleotides allows us to detect the products at 260 nm via a UV detector even though the T[c,s]T and TA* photoproducts exhibit little absorbance at this wavelength (6, 35). Implications for Enzymology. Because mass spectrometry is sensitive and MS/MS can provide structure information, mass spectrometry can have an impact on understanding enzymatic mechanisms by allowing enzymatic reaction products to be easily identified and quantified. A recent X-ray cocrystal structure of nuclease P1 and dithiophosphorylated d(ATTT) indicates that stacking and hydrogen-bonding interactions with the base 5′ to the phosphodiester bond that is to be cleaved are needed for substrate binding and recognition (30). This is in contrast to an earlier crystal-soaking experiment of nuclease P1 with dA‚P(S)‚dA. The results of that study suggest that the base 3′ to the phosphate to be cleaved is important for substrate recognition (31). Digestion experiments with dinucleotide substrates lacking either the 3′ or 5′ base or having modified bases at these positions (36) also support the conclusion that the 5′ base is important for substrate recognition (32). The inhibitory effect of modifications to the structure of the nucleotide

P1 Digestion and MS/MS for DNA Photoproducts

5′ to the cleavage site is also seen in the digestion of (5′S)cycloAdo (27)- and 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine-containing ODNs (26). In our case, cleavage of the phosphodiester bond 3′ to the cis-syn, [6-4], and TA* photoproducts is inhibited, which is consistent not only with the X-ray results reported more recently (30) but also with the results of Weinfeld et al. (33) with cis-syn dimer-containing trinucleotides, although the hydrolysis of the intradimer phosphodiester bond of d(T[c,s]TT) was also observed in the latter study. We did not observe this kind of hydrolysis as no peaks corresponding to [M + H2O] were seen in the MALDI-TOF spectra of the photoproduct-containing trinucleotide digestion products. Examination of the most recent crystal structure of nuclease P1 reveals that the nucleotide 5′ to the cleavage site is bound to the enzyme by way of a deep pocket. This would explain why neither nucleobase of the dinucleotide photoproduct can bind at this site. As a result, the 3′ and intra-photoproduct phosphodiester bonds are not targets for hydrolysis, and the unmodified base 3′ to the photodamage site is retained in the digestion product. By taking advantage of the structure information of the digestion products obtained by mass spectrometry, we are able not only to determine the structure of the photoproduct but also to obtain information about the mechanism of enzymesubstrate recognition and reaction.

Conclusion Using the method, we can establish rapidly the types of photoproducts from their characteristic fragmentation patterns in MS/MS. This capability should be the basis of an appropriate tool for the discovery of new photoproducts and for other detailed studies of DNA photochemistry. The base immediately 3′ to the photoproduct can also be determined from the molecular weight of the nuclease P1 digestion product, and this information may allow us to do nearest-neighbor analysis and identify hot spots for photodamage in cellular DNA (11). Compared with the previous structural approach developed in this laboratory (18, 20), the present method can deal with longer DNA strands. The enzymatic digestion procedure is nondestructive with respect to the photodamaged site, making this procedure more suitable for acid labile photoproducts, such as those involving TA*, than the conventional assay, which uses formic acid hydrolysis (19). The MS method is also sensitive, requiring small picomole quantities of sample for ESI-MS/MS or MALDIPSD. In the future, we intend to apply the method to the search for new DNA photoproducts and to the study of photomodified DNA in real biological samples. For those samples, the product variety stemming from a combination of different nucleobases and photoproducts can make the nuclease P1 digestion products complex; however, capillary HPLC should provide sufficient separation capability (14), and that coupled with ESI-MS and MS/ MS should provide structure information. We also intend to use the mass spectrometry approach as a tool for studying the mechanism of DNA photodamage repair and replication. The method that is described, however, should also be generally useful for studying all types of DNA modifications, and for determining the mechanism of a variety of DNA processing enzymes.

Acknowledgment. The research was supported by grants from the National Center for Research Resources

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of the NIH (P41RR00954), from the NIH (Grants CA40463 and CA49210), and from Alchemy International (Middleburg, VA).

Note Added in Proof The considerations discussed in this article do not apply for photomodifications at the end of an ODN. If the modification is at the 3′ end, a dinucleotide diphosphate is expected (24). If the modification is at the 5′ end, a trinucleotide diphosphate is produced.

References (1) Basu, A. K., and Essigmann, J. M. (1988) Site-specifically modified oligodeoxynucleotides as probes for the structural and biological effects of DNA-damaging agents. Chem. Res. Toxicol. 1, 1-18. (2) Grossman, D., and Leffell, D. J. (1997) The molecular basis of nonmelanoma skin cancer: new understanding. Arch. Dermatol. 133, 1263-1270. (3) Muller, L., Kasper, P., Kersten, B., and Zhang, J. (1998) Photochemical genotoxicity and photochemical carcinogenesisstwo sides of a coin? Toxicol. Lett. 102-103, 383-387. (4) Pfeifer, G. P. (1997) Formation and processing of UV photoproducts: effects of DNA sequence and chromatin environment. Photochem. Photobiol. 65, 270-283. (5) Jagger, J. (1976) in Photochemistry and Photobiology of Nucleic Acids (Wang, S. Y., Ed.) Vol. 2, p 184, Academic Press, New York. (6) Bose, S. N., Davies, R. J. H., Sethi, S. K., and McCloskey, J. A. (1983) Formation of an Adenine-thymine Photoadduct in the Deoxydinucleoside Monophosphate d(TpA) and in DNA. Science 220, 723-725. (7) Zhao, X., Nadji, S., Kao, J. L.-F., and Taylor, J.-S. (1996) The structure of d(TpA)*, the major photoproduct of thymidylyl-(3′5′)-deoxyadenosine. Nucleic Acids Res. 24, 1554-1560. (8) Cadet, J., and Vigny, P. (1990) in Bioorganic Photochemistry (Morrison, H., Ed.) Vol. 1, p 1, John Wiley, New York. (9) Taylor, J.-S. (1995) Sunlight and skin cancer. IUPAC 67, 183190. (10) Zhao, X., and Taylor, J.-S. (1996) Mutation spectra of TA*, the major photoproduct of thymidylyl-(3′-5′)-deoxyadenosine, in Escherichia coli under SOS conditions. Nucleic Acids Res. 24, 15611565. (11) Weinfeld, M., Liuzzi, M., and Paterson, M. C. (1989) Enzymatic analysis of isomeric trithymidylates containing ultraviolet lightinduced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase. J. Biol. Chem. 264, 6364-6370. (12) Randerath, K., Reddy, M. V., and Gupta, R. C. (1981) 32P-labeling test for DNA damage. Proc. Natl. Acad. Sci. U.S.A. 78, 61266129. (13) Bykov, V. J., and Hemminki, K. (1996) Assay of different photoproducts after UVA, B and C irradiation of DNA and human skin explants. Carcinogenesis 17, 1949-1955. (14) Bykov, V. J., Kumar, R., Forsti, A., and Hemminki, K. (1995) Analysis of UV-induced DNA photoproducts by 32P-postlabelling. Carcinogenesis 16, 113-118. (15) Bykov, V. J., and Hemminki, K. (1995) UV-induced photoproducts in human skin explants analysed by TLC and HPLC-radioactivity detection. Carcinogenesis 16, 3015-3019. (16) Bykov, V. J., Jansen, C. T., and Hemminki, K. (1998) High levels of dipyrimidine dimers are induced in human skin by solarsimulating UV radiation. Cancer Epidemiol. Biomarkers Prev. 7, 199-202. (17) Bykov, V. J., Marcusson, J. A., and Hemminki, K. (1998) Ultraviolet B-induced DNA damage in human skin and its modulation by a sunscreen. Cancer Res. 58, 2961-2964. (18) Vollmer, D., Zhao, X., Taylor, J.-S., and Gross, M. L. (1997) Structure determination of isomeric hexadeoxynucleotide photoproducts by high-field NMR and fast atom bombardment/tandem mass spectrometry. Int. J. Mass Spectrom. Ion Processes 165/166, 487-496. (19) Cadet, J., Gentner, N. E., Rozga, B., and Paterson, M. C. (1983) Rapid quantitation of ultraviolet-induced thymine-containing dimers in human cell DNA by reversed-phase high-performance liquid chromatography. J. Chromatogr. 280, 99-108. (20) Wang, Y., Taylor, J.-S., and Gross, M. L. (1999) Differentiation of isomeric photomodified oligodeoxynucleotides by fragmentation of ions produced by matrix-assisted laser desorption ionization and electrospray ionization. J. Am. Soc. Mass Spectrom. 10, 329338.

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(21) Janning, P., Schrader, W., and Linscheid, M. (1994) A new mass spectrometric approach to detect modifications in DNA. Rapid Commun. Mass Spectrom. 8, 1035-1040. (22) Shishido, K., and Ando, T. (1982) in Nucleases (Linn, S., and Roberts, R. J., Eds.) pp 155-209, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (23) Cadet, J., and Weinfeld, M. (1993) Detecting DNA damage. Anal. Chem. 65, 675A-682A. (24) Wu, H., Morgan, R. L., and Aboleneen, H. (1998) Characterization of labelled oligonucleotides using enzymatic digestion and tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 9, 660-667. (25) Gasparutto, D., Ravanat, J.-L., Gerot, O., and Cadet, J. (1998) Characterization and chemical stability of photooxidized oligonucleotides that contain 2,2-diamino-4-[(2-deoxy-D-erythro-pentofuranosyl)amino-5(2H)-oxazolone. J. Am. Chem. Soc. 120, 1028310286. (26) Romieu, A., Gasparutto, D., Molko, D., Ravanat, J.-L., and Cadet, J. (1999) Synthesis of oligonucleotides containing the (4R) and (4S) diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2′-deoxyguanosine. Eur. J. Org. Chem., 49-56. (27) Romieu, A., Gasparutto, D., Molko, D., and Cadet, J. (1998) Sitespecific introduction of (5′S)-5′,8-cyclo-2′-deoxyadenosine into oligodeoxyribonucleotides. J. Org. Chem. 63, 5245-5249. (28) Deforce, D. L., Ryniers, F. P., van den Eeckhout, E. G., Lemiere, F., and Esmans, E. L. (1996) Analysis of DNA adducts in DNA hydrolysates by capillary zone electrophoresis and capillary zone electrophoresis-electrospray mass spectrometry. Anal. Chem. 68, 3575-3584. (29) Deforce, D. L., Lemiere, F., Hoes, I., Millecamps, R. E., Esmans, E. L., De Leenheer, A., and Van den Eeckhout, E. G. (1998)

Wang et al.

(30)

(31) (32)

(33)

(34) (35) (36)

Analysis of the DNA adducts of phenyl glycidyl ether in a calf thymus DNA hydrolysate by capillary zone electrophoresiselectrospray mass spectrometry: evidence for phosphate alkylation. Carcinogenesis 19, 1077-1086. Romier, C., Dominguez, R., Lahm, A., Dahl, O., and Suck, D. (1998) Recognition of single-stranded DNA by nuclease P1: highresolution crystal structures of complexes with substrate analogs. Proteins 32, 414-424. Volbeda, A., Lahm, A., Sakiyama, F., and Suck, D. (1991) Crystal structure of Penicillium citrinum P1 nuclease at 2.8 Å resolution. EMBO J. 10, 1607-1618. Weinfeld, M., Soderlind, K. J., and Buchko, G. W. (1993) Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA. Nucleic Acids Res. 21, 621-626. Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) Enzymatic analysis of isomeric trithymidylates containing ultraviolet lightinduced cyclobutane pyrimidine dimers. I. Nuclease P1-mediated hydrolysis of the intradimer phosphodiester linkage. J. Biol. Chem. 264, 6355-6363. Ulrich, J., Cadet, J., Becchi, M., and Fraisse, D. (1989) in Advances in Mass Spectrometry (Longevialle, P. L., Ed.) pp 494-495, Heyden & Sons, London. Setlow, R. B. (1966) Cyclobutane-type pyrimidine dimers in polynucleotides. Science 153, 379-386. Weinfeld, M., Liuzzi, M., and Paterson, M. C. (1989) Selective hydrolysis by exo- and endonucleases of phosphodiester bonds adjacent to an apurinic site. Nucleic Acids Res. 17, 3735-3745.

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