Polymerase Incorporation and Miscoding Properties of 5-Chlorouracil

Jan 28, 2010 - Department of Basic Sciences, School of Medicine, Loma Linda UniVersity, Loma Linda, California 92350. ReceiVed August 25, 2009...
0 downloads 0 Views 1MB Size
Download PDF
740

Chem. Res. Toxicol. 2010, 23, 740–748

Polymerase Incorporation and Miscoding Properties of 5-Chlorouracil Cherine H. Kim, Agus Darwanto, Jacob A. Theruvathu, Jason L. Herring, and Lawrence C. Sowers* Department of Basic Sciences, School of Medicine, Loma Linda UniVersity, Loma Linda, California 92350 ReceiVed August 25, 2009

Inflammation-mediated hypochlorous acid (HOCl) can damage DNA, DNA precursors, and other biological molecules, thereby producing an array of damage products such as 5-chlorouracil (ClU). In this study, we prepared and studied 5-chloro-2′-deoxyuridine (CldU) and ClU-containing oligonucleotide templates. We demonstrate that human K-562 cells grown in culture with 10 µM CldU incorporate substantial amounts of CldU without significant toxicity. When in the template, ClU residues pair with dATP but also with dGTP, in a pH-dependent manner with incorporation by human polymerase β, avian myeloblastosis virus reverse transcriptase (AMV-RT), and Escherichia coli Klenow fragment (exo-) polymerase. The enhanced miscoding of ClU is attributed to the electron-withdrawing 5-chlorine substituent that promotes the formation of an ionized ClU-G mispair. When mispaired with G, ClU is targeted for removal by human glycosylases. The formation, incorporation, and repair of ClU could promote transition mutations and other forms of heritable DNA damage. Introduction Inflammation and cancer have been linked in numerous studies (1-3), yet the mechanistic basis for this relationship remains unknown. Components of innate immunity generate highly reactive molecules that have potent antimicrobial activity; however, these chemically reactive species can cause collateral damage to host molecules including proteins, lipids, and nucleic acids. Among these reactive species are superoxide, hydrogen peroxide, and HOCl, the latter generated by myeloperoxidase from activated neutrophils (4-9). Among the known nucleic acid reaction products resulting from inflammation-mediated reactive species are oxidized and halogenated DNA bases, including 5-chlorouracil (ClU) (5-7). Of the halogenated uracils, ClU is arguably the most likely product of inflammationmediated damage. 5-Fluorouracil (FU) and 5-iodouracil (IU) have yet to be reported under physiological conditions. 5-Bromouracil (BrU) is a potential byproduct of inflammation from eosinophil peroxidase-derived HOBr (5); however, since the number of neutrophils in human blood is substantially larger than that of eosinophils, we anticipate that physiological ClU formation exceeds that of BrU. Although ClU is perhaps the most likely halogenated base to form in mammalian tissues, little is currently known about the biological impact of this damaged base. Substantial work has been published previously on the similar halogenated analogues 5-fluorouracil (FU) and 5-bromouracil (BrU) (10-18). FU is a chemotherapy agent used in the treatment of several human malignancies, whereas BrU is a thymine mimic and mutagenic base analogue. The distinct differences in the biological properties of FU and BrU can be attributed in part to the different sizes of the 5-fluoro and 5-bromo substituents. The smaller size of the 5-substituent of the corresponding 2′-deoxynucleotide analogue of FU (FdUMP) inhibits thymidylate synthase (TS), diminishing the TMP needed * To whom correspondence should be addressed. Tel: 909-558-4480. Fax: 909-558-4035. E-mail: [email protected].

for DNA replication (19). Furthermore, both uracil and FU incorporated into DNA as a consequence of TS inhibition result in DNA glycosylase-mediated DNA fragmentation (19, 20). Glycosylases are known to distinguish uracil and FU from BrU and thymine upon the basis of the size of the 5-substituent (21-27). Since the 5-chloro group of ClU is intermediate in size between fluoro and bromo, the biological properties of ClU could overlap with both FU and BrU. While it is known that 5-chloro-2′-deoxyuridine (CldU) added to culture media is metabolized and incorporated into DNA as shown by antibody binding (28-32), CldU induces senescence, toxicity, and sisterchromatid exchanges by as yet unknown mechanisms (33-36). The electronic-inductive properties of all three halogens, F, Cl, and Br, are similar in that they withdraw electron density from the pyrimidine ring, increasing the acidity of both the N1 and N3 protons (37, 38). An electron-withdrawing substituent in the 5-position of a pyrimidine deoxynucleoside also weakens the glycosidic bond and renders the corresponding deoxynucleoside more susceptible to cleavage by DNA repair glycosylases (22-27). While ClU residues in DNA can be cleaved by glycosylases, the efficiency of repair appears to be highly context-dependent (25). All three halogenated bases form base pairs with adenine in duplex DNA in a configuration similar to that of a normal T-A base pair (13-15, 39). When mispaired with guanine, all three halogenated base pairs undergo a pHdependent transition from a neutral wobble to ionized base pair in a configuration approaching that of a Watson-Crick base pair (16-18, 40). Previous studies have examined the polymerase coding properties of FU and BrU and have established that the mispairing of both analogues increases with pH, consistent with the role of base ionization in miscoding (41, 42). In this study, we have examined the polymerase-directed coding properties of the ClU analogue as a template base in an oligodeoxyribonucleotide with three polymerases: human polymerase β, avian myeloblastosis virus reverse transcriptase, and Escherichia coli Klenow fragment (exo-). These three poly-

10.1021/tx900302j  2010 American Chemical Society Published on Web 01/28/2010

Polymerase Miscoding of 5-Chlorouracil

Figure 1. Schematic diagram of CldU and CldU-phosphoramidite.

Figure 2. Template and primer sequences. Sequences for studies of (A) incorporation of dATP or dGTP opposite template ClU or T residue. (B) Repair by hSMUG1 glycosylase.

merases comprise a group of model polymerases from eukaryotic, viral, and prokaryotic origin. Using mass spectrometry, we have also examined the incorporation of 5-chloro-2′deoxyuridine (CldU) into the DNA of replicating mammalian cells and the repair of ClU by the human glycosylase, SMUG1, when paired with adenine or mispaired with guanine. The results reported here may, in part, explain some of the biological properties of ClU incorporation into DNA.

Materials and Methods Synthesis and Characterization of CldU, ClU Phosphoramidite and Oligonucleotides Containing ClU. Synthesis of CldU. Commercially available 2′-deoxyuridine or dU (Chem-Impex International, Wood Dale, IL) was converted to 5-chloro-2′deoxyuridine (CldU), as diagramed in Figure 1, according to the method published by Kumar et al. (43). The product CldU was characterized by NMR and mass spectrometry. Synthesis of ClU-Phosphoramidite. The phosphoramidite of CldU was prepared by standard methods, as previously described. CldU was dried with anhydrous pyridine and converted to the 5-dimethyoxytrityl derivative. The corresponding 3′-cyanoethyldiisopropylaminophosphoramidite was prepared using established procedures (44). Synthesis of Oligonucleotides Containing ClU. Oligonucleotide resins and phosphoramidites of the normal DNA bases were obtained from Glen Research (Sterling, VA). Oligonucleotide synthesis was conducted with either a Pharmacia gene assembler (GE Healthcare Bio-Sciences, Piscataway, NJ) or an Expedite oligonucleotide synthesizer from Applied Biosystems (Foster City, CA). Oligonucleotides containing ClU were deprotected with concentrated aqueous ammonia at room temperature for 24 h and purified by HPLC. Phosphoramidites and precursors as well as oligonucleotides containing ClU were characterized by NMR and mass spectrometry as previously described (9, 39, 40). Oligonucleotide sequences utilized in this study are shown in Figure 2. Incorporation of CldU into the DNA of K-562 Cells. Growth of Cells in CldU-Containing Media. Human erythroleukemia K-562 cells obtained from ATCC (CLL-243) were grown at 37 °C in a humidified incubator at 5% CO2 in RPMI-1640 media (Cellgro, Manassas, VA). Media was supplemented with 10% fetal bovine serum (Gibco-Invitrogen, Carlsbad, CA) and 1% L-glutamine-penicillin-streptomycin solution. Cells were initially seeded at 1.0 × 105 cells/mL and treated with either 10 µM CldU or 10 µM thymidine (negative control) for 63 h or two cell doublings (45). Cells were counted by trypan blue exclusion, pelleted by centrifugation (500g), and washed with 10 mL of sterile PBS.

Chem. Res. Toxicol., Vol. 23, No. 4, 2010 741 DNA Extraction and GC/MS Analysis. DNA was extracted from pelleted cells using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). The amount of DNA in samples was determined by measuring the ratio of UV absorbance at 260 and 280 nm. Samples containing approximately 1 µg of DNA were dried under reduced pressure and resuspended in 88% formic acid in sealed vials and heated at 140 °C for 40 min. Formic acid was removed under reduced pressure, and the residue containing purine and pyrimidine free bases was derivatized in 20 µL of anhydrous acetonitrile and 20 µL of MTBSTFA with 1% TBDMCS (Pierce, Rockford, IL) in sealed vials at 140 °C for 40 min. Samples were injected into the GC/MS (Hewlett-Packard, Palo Alto, CA) in splitless mode. Mass spectra were collected in the selected ion mode, monitoring the M-57 fragment ion for each pyrimidine and purine examined (45). The retention times (min) for the silylated DNA bases were thymine (12.01), 5-chlorouracil (12.33), cytosine (12.57), 5-methylcytosine (12.68), adenine (14.41), and guanine (15.03). A standard curve was established using authentic standards between ClU and T comparing relative concentrations with relative peak areas (Figure S1, Supporting Information). Polymerase Kinetic Studies for ClU Miscoding As a Function of pH Derived from Gel Electrophoretic Studies. Polymerase Incorporation Assays. Human polymerase β (pol β) was obtained from Enzymax (Lexington, KY), and AMV-RT and exonuclease-deficient Klenow fragment (exo-) polymerase were obtained from New England Biolabs (Ipswich, MA). For the polymerase incorporation assays, the primers were 5′-32P-end labeled by T4 polynucleotide kinase (New England Biolabs) with [γ-32P]adenosine triphosphate (MP Biomedicals, Costa Mesa, CA) under conditions recommended by the enzyme supplier. The labeled oligonucleotides were purified using G25 Sephadex columns (Roche Applied Science, Indianapolis, IN). A 2-fold excess of the complementary template strand was then added to the labeled primer mixture, incubated at 95 °C for 5 min, and allowed to cool to room temperature gradually to create the oligo-primer duplex. Optimal buffer conditions were used for each polymerase examined. For polymerase incorporation assays with pol β, 50 nM of labeled substrate was incubated with 63 nM pol β, 15 µM of the complementary TTP (running start), and increasing concentrations of the target site dNTP in 1× buffer (20 mM NaCl, 20 mM KCl, 50 mM Tris-HCl, 10 mM MgCl2, 2 mM dithiothreitol, 20 µg/mL BSA, and 1% glycerol, pH 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 at 25 °C) in a total volume of 20 µL at 37 °C for 30 min. For assays with AMV-RT, 33.05 nM of the labeled substrate was incubated with 2.83 nM AMV-RT, 100 µM of the complementary running start dNTP, and increasing concentrations of the target site dNTP in 1× buffer (75 mM NaCl, 60 mM Tris-HCl, 8 mM MgCl2, and 0.5 mM dithiothreitol, pH 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 at 25 °C) in a total volume of 20 µL at 37 °C for 30 min. For assays with Klenow polymerase, 50 nM of labeled substrate was incubated with 10 nM Klenow, 500 nM of the complementary running start dNTP, and increasing concentrations of the target site dNTP in 1× buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol, pH 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 at 25 °C) in a total volume of 20 µL at 37 °C for 30 min. Reactions were stopped using equal volumes of Maxam-Gilbert loading buffer (98% formamide, 0.01 M EDTA, 1 mg/mL xylene cyanole, and 1 mg/ mL bromophenol blue). The reaction products were electrophoresed on 20% denaturing polyacrylamide gels containing 8 M urea and quantified using a phosphorimager and ImageQuant 5.2 software (Molecular Dynamics, GE Healthcare Bio-Sciences). The incorporation of two complementary dNTPs in this running start model confirms the viability of the 3′ end of the primer for extension. The third position of incorporation, the target site, is the site of interest for kinetic measurements. The relative nucleotide insertion at the template target site was determined by calculating the ratio of band intensity of the primer extended at the target site compared with the band intensity of primer extended to a site just prior to the target. When using high concentrations of the correct running start nucleotide, TTP, the primer is rapidly extended to the target site, and incorporation at the target site becomes rate-limiting.

742

Chem. Res. Toxicol., Vol. 23, No. 4, 2010

Kim et al.

Table 1. Kinetic Parameter Values (kcat, Km) of Incorporation of dATP and dGTP against a Template ClU Residue with Pol β, AMV-RT, and Klenow at pH 7.5 pol β dATP:ClU dGTP:ClU

kcat Km kcat Km

(s-1) (M) (s-1) (M)

1.20 1.65 4.95 2.20

× × × ×

10-04 10-07 10-05 10-04

( ( ( (

1.57 8.76 8.21 1.31

AMV-RT × × × ×

10-05 10-08 10-06 10-04

3.75 3.17 8.30 3.90

× × × ×

10-02 10-07 10-03 10-04

( ( ( (

4.81 1.57 1.38 2.17

Klenow × × × ×

10-03 10-07 10-03 10-04

1.06 6.39 2.21 2.67

× × × ×

10-03 10-09 10-03 10-05

( ( ( (

4.38 1.44 2.05 9.79

× × × ×

10-05 10-09 10-04 10-06

Determination of kcat and Km at pH 7.5. The values of kcat and Km for the incorporation of dNTP against a template ClU residue were determined using the polymerase incorporation assays described above at pH 7.5. A series of reaction mixtures containing increasing concentrations of dNTP were incubated for 0, 0.5, 2, 5, 15, 30, or 120 min. The concentration of extended product formed by nucleotide insertion at the template target was determined by calculating the ratio of band intensity of the primer extended at the target site with the band intensity of the primer extended to a site just prior to the target. Initial velocities were evaluated by plotting product concentration versus time. The kcat and Km values were obtained by a nonlinear least-squares fit of velocity versus dNTP concentration. Determination of Kinetic Parameters of Nucleotide Incorporation as a Function of pH. The relationship between enzyme velocity and the kinetic parameters Km and Vmax can be written as V0 ) (Vmax × [S])/(Km + [S]). As presented by Goodman and colleagues (46, 47), velocity can be used to estimate Vmax/Km when Km > [S]. The values of Vmax/Km were determined at fixed substrate concentration, [S], and the substrate concentration used in these measurements was less that 15% of the Km value for each polymerase shown in Table 1. For assays with pol β examining the incorporation of dATP or dGTP opposite ClU and T, the nucleotide concentrations used were 25 nM and 30 µM, respectively. For assays with AMV-RT examining the incorporation of dATP opposite ClU and T, the dATP concentrations used were 10 nM and 30 nM, respectively; for the incorporation of dGTP opposite ClU and T, the dGTP concentration was 60 µM. For assays with Klenow examining the incorporation of dATP or dGTP opposite ClU and T, the nucleotide concentrations used were 1 nM and 4 µM, respectively. Using the polymerase incorporation assays described above, the primer was extended by the addition of the complementary nucleotide up to the T or ClU target site. The incorporation opposite the target base was measured as a function of the complementary or noncomplementary nucleotide. The relative velocity of nucleotide insertion at the template target site was determined by calculating the ratio of band intensity of the primer extended to the target site compared with the band intensity of the primer extended to a site just prior to the target. The average Vmax/Km value and standard error for each polymerase was determined from three independent data sets. Examination of hSMUG1 Removal of ClU. The glycosylase studies were performed using human single-stranded selective monofunctional uracil-DNA glycosylase (hSMUG1). hSMUG1 was cloned and purified by our lab (27). One hundred picomoles of each self-cDNA substrate shown in Figure 2B (T-G, ClU-A, and ClU-G) was annealed separately in 20 mM Tris-HCl at pH 8.0, 1 mM EDTA, 1 mM DTT, 50 mM NaCl, and 0.1 mg/mL BSA. A typical reaction was done by incubating with 1.4 nmol of recombinant hSMUG1 at 37 °C for 24 h. The sample was then desalted for MALDI-TOF analysis with a Biospin 6 column and a cation exchange column containing AG50W-X8 beads on the H+ form (Bio-Rad, Hercules, CA). MALDI-TOF experiments were performed with a Bruker Autoflex time-of-flight mass spectrometer operated in positive ion and reflectron modes (48). Kinetic studies of ClU-A cleavage by hSMUG1 were also performed with 5′-32P-end labeled substrates and gel electrophoresis, as described previously (27).

characterized by NMR spectroscopy and mass spectrometry as previously described (9, 39, 40). CldU was also converted to the corresponding 5′-dimethoxytrityl-3′-phosphoramidite derivative and inserted into synthetic oligonucleotides (Figure 2). The composition of ClU-containing oligonucleotides was verified by GC/MS analysis following formic acid hydrolysis. The mass spectrum of a chlorine-containing molecule is easily discerned by the characteristic appearance of the M and M + 2 isotopes (Figure 3C). Incorporation of CldU into the DNA of Replicating Mammalian Cells. We next examined the incorporation of CldU into the DNA of human K-562 cells grown in CldU containing culture media. The concentration of CldU in the media was 10 µM, and cells were grown for two cell doublings. Cells were harvested, and the DNA was extracted and separated from low molecular weight metabolites. The extracted DNA was hydrolyzed in formic acid, and the liberated free bases were

Results

Figure 3. GC/MS analysis of DNA bases from DNA extracted from K-562 cells following growth in dT or CldU containing media. Total ion chromatogram of DNA bases in K-562 cells exposed to (A) 10 µM dT or (B) 10 µM CldU. (C) Mass spectrum of the TBDMS derivative of ClU.

Preparation and Characterization of CldU and Oligonucleotides Containing a ClU Residue. In this study, commercially available dU was converted to CldU (Figure 1) and

Polymerase Miscoding of 5-Chlorouracil

Chem. Res. Toxicol., Vol. 23, No. 4, 2010 743

Table 2. Kinetic Parameter Values (Vmax/Km) of Incorporation of dATP against a Template ClU or T Residue and Misincorporation of dGTP against a Template ClU and T Residue with Pol β, AMV-RT, and Klenow Determined from Three Independent Data Sets Vmax /Km (s-1) pH dATP:ClU

dATP:T

dGTP:ClU

dGTP:T

6.5 7.0 7.5 8.0 8.5 9.0 9.5 6.5 7.0 7.5 8.0 8.5 9.0 9.5 6.5 7.0 7.5 8.0 8.5 9.0 9.5 6.5 7.0 7.5 8.0 8.5 9.0 9.5

pol β 1.18 2.01 3.15 5.09 5.61 4.06 3.57 1.33 2.78 3.50 5.76 5.27 4.11 2.94 1.11 2.01 3.56 1.01 3.21 4.65 5.56 4.91 7.84 9.70 1.52 1.77 5.20 1.08

× × × × × × × × × × × × × × × × × × × × × × × × × × × ×

10-05 10-05 10-05 10-05 10-05 10-05 10-05 10-05 10-05 10-05 10-05 10-05 10-05 10-05 10-08 10-08 10-08 10-07 10-07 10-07 10-07 10-09 10-09 10-09 10-08 10-08 10-08 10-07

( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

6.75 5.99 3.55 2.63 5.78 3.92 1.04 4.72 4.29 8.55 1.04 1.09 1.56 1.02 4.57 1.80 4.87 1.16 1.69 1.12 1.56 1.06 9.51 2.23 2.62 1.81 6.47 1.20

AMV-RT × × × × × × × × × × × × × × × × × × × × × × × × × × × ×

10-06 10-06 10-06 10-06 10-06 10-06 10-06 10-06 10-06 10-06 10-05 10-05 10-05 10-05 10-10 10-09 10-09 10-08 10-08 10-08 10-08 10-09 10-10 10-09 10-09 10-09 10-09 10-08

converted to their TBDMS derivatives and analyzed by GC/ MS. Peaks corresponding to the normal bases C, T, A, G, and 5-methylcytosine (5mC) were observed in the total ion chromatograms shown in Figure 3A and B. An additional peak was observed in the chromatogram of CldU treated cells (Figure 3B) at 12.33 min, the mass spectrum of which (Figure 3C) corresponds to 5-chlorouracil. The T content in the DNA from CldU treated cells (Figure 3B) is decreased in comparison to that of the control cells (Figure 3A), whereas the ClU content increases proportionately. The similar size of the T and ClU peaks (Figure 3B) indicates that the DNA of the cultured cells contain similar amounts of T and ClU. Upon the basis of a standard curve comparing peak volumes with a relative concentration of ClU and T (Figure S1, Supporting Information), the DNA extracted from the cells contained a mole ratio of ClU to T of 0.89 ( 0.02. Under the culture conditions used here, the percent viable cells in the CldU-treated culture was approximately 95%, as compared to thymidine-treated controls. These data reveal that a substantial amount of CldU is incorporated into the DNA of growing mammalian cells with minimal apparent toxicity over the time period examined here. Incorporation of dATP Opposite a Template ClU Residue. The capacity of ClU in synthetic oligonucleotides to serve as a template for polymerase-directed incorporation of dATP was then investigated. The template-primer complexes used in this study, containing ClU or T, are shown in Figure 2A. The incorporation of the normal triphosphate, dATP, opposite template ClU and T residues is demonstrated using the same gel assay described above. The Km and kcat for the insertion of dATP opposite template ClU at pH 7.5 are given in Table 1. In order to estimate the impact of increasing pH on the incorporation of dATP opposite template ClU and T residues, Vmax/Km was estimated at selected values of pH from 6.5 to 9 or 9.5 by measuring Vo/[S] at a fixed [S], where [S] was no more than 15% of Km for each enzyme as described in Materials

2.42 2.83 2.80 1.97 1.03 7.59 N/A 1.82 2.00 2.17 1.49 8.77 5.50 N/A 1.10 1.73 4.72 8.60 1.04 1.08 N/A 1.33 1.72 2.79 4.48 5.91 2.06 N/A

Klenow

× × × × × ×

10-04 10-04 10-04 10-04 10-05 10-05

( ( ( ( ( (

2.74 9.02 3.25 2.63 1.55 7.73

× × × × × ×

10-05 10-06 10-05 10-05 10-05 10-06

× × × × × ×

10-04 10-04 10-04 10-04 10-05 10-05

( ( ( ( ( (

1.11 1.05 4.17 1.46 5.83 7.48

× × × × × ×

10-05 10-05 10-06 10-05 10-06 10-06

× × × × × ×

10-08 10-08 10-08 10-08 10-07 10-07

( ( ( ( ( (

2.07 2.93 1.34 3.89 1.49 4.90

× × × × × ×

10-09 10-09 10-09 10-09 10-09 10-09

× × × × × ×

10-08 10-08 10-08 10-08 10-08 10-08

( ( ( ( ( (

1.21 1.14 4.82 2.77 2.47 6.23

× × × × × ×

10-09 10-09 10-09 10-09 10-09 10-09

8.38 9.60 1.33 1.70 1.23 9.62 N/A 5.98 1.29 1.76 2.14 1.53 1.05 N/A 5.93 7.39 1.21 2.86 3.90 4.61 N/A 2.64 4.72 8.00 1.77 2.22 2.59 N/A

× × × × × ×

10-05 10-05 10-04 10-04 10-04 10-05

( ( ( ( ( (

1.42 1.59 1.82 1.27 2.05 1.24

× × × × × ×

10-05 10-06 10-05 10-05 10-05 10-05

× × × × × ×

10-05 10-04 10-04 10-04 10-04 10-04

( ( ( ( ( (

4.75 3.08 1.68 2.58 2.36 1.55

× × × × × ×

10-06 10-05 10-05 10-05 10-05 10-05

× × × × × ×

10-08 10-08 10-07 10-07 10-07 10-07

( ( ( ( ( (

3.12 3.85 2.32 2.69 2.68 4.45

× × × × × ×

10-08 10-09 10-08 10-08. 10-08 10-08

× × × × × ×

10-08 10-08 10-08 10-07 10-07 10-07

( ( ( ( ( (

1.65 1.42 1.41 1.10 1.43 9.27

× × × × × ×

10-08 10-09 10-08 10-08 10-08 10-09

and Methods. These data are presented in Table 2 and plotted in Figure S2 (Supporting Information). These data show that the polymerase-mediated incorporation of dATP opposite template ClU and T does vary with increasing pH; however, the observed incorporation of dATP opposite template ClU does not differ substantially from incorporation opposite template T at any value of pH for any of the three polymerases examined. These data confirm that the template properties of ClU and T are similar with respect to dATP incorporation. Misincorporation of dGTP Opposite Template ClU by DNA Polymerases. The kinetic parameters for the incorporation of dGTP opposite template ClU at pH 7.5 were determined as shown in Table 1. When comparing the correct incorporation of dATP opposite template ClU versus the incorrect incorporation of dGTP, the observed Km for incorrect incorporation is 3 orders of magnitude higher than that for correct incorporation. The value of kcat for incorrect base pair formation declines by approximately 1 order of magnitude. Similar results were obtained with the three model polymerases examined. The misincorporation of dGTP opposite template ClU and T was then investigated as functions of both dGTP concentration and solution pH. Gel electrophoretic data obtained with pol β is shown in Figure 4. Corresponding gels for AMV reverse transcriptase and Klenow polymerase are shown in Supporting Information (Figures S3 and S4). These data visually illustrate the difference between the misincorporation of dGTP opposite template ClU and T with increasing pH. With all three polymerases, the misincorporation of dGTP opposite both ClU and T increases with increasing dGTP concentration and increasing pH; however, the impact of increasing pH is much more apparent with dGTP incorporation opposite ClU than with template T. The misinsertion efficiency of dGTP opposite template T and ClU residues, Vmax/Km, was measured as described above for dATP as a function of pH, and the results are recorded in Table

744

Chem. Res. Toxicol., Vol. 23, No. 4, 2010

Kim et al.

Figure 4. Gel electrophoretic analysis of the incorporation by pol β of dGTP against template ClU and T residues as a function of solution pH. (A) Template ClU. (B) Template T. A labeled primer is extended by the incorporation of two T deoxynucleotides (TTP is present at a saturating concentration of 15 µM) to reach the template target site ClU or T. Lane 1 includes the primer only, while dGTP is present in the following concentrations: lane 2, 0 µM; lane 3, 15 µM; lane 4, 30 µM; lane 5, 60 µM; lane 6, 125 µM; lane 7, 250 µM; lane 8, 1000 µM; lane 9, 3000 µM. Template sequences corresponding to Figure 2A and the incorporated bases are shown on the right side of the figure.

2 and plotted in Figure 5. For pol β and Klenow, the misincorporation opposite both ClU and T increases continuously with increasing pH, whereas with AMV-RT, the misinsertion of dGTP opposite T shows an apparent optimum of pH 8.5. Both the visual presentation of the gel data shown in Figures 4, S3 (Supporting Information), and S4 (Supporting Information) and the graphical presentation of the measured Vmax/Km values show that the misincorporation of dGTP opposite template ClU increases with increasing pH more rapidly than that with template T. The increased misinsertion of dGTP opposite ClU with increasing pH is consistent with the formation of an ionized base pair as shown in Figure 6. The pH-dependent insertion frequency of dGTP opposite template ClU (Table 2) was normalized to the pH-dependence of dATP insertion opposite template ClU (Table 2) to generate the misinsertion parameter, fins:

fins ) (Vmax /Km)ClU-G /(Vmax /Km)ClU-A

(1)

To determine if a relationship exists between the misinsertion of dGTP and the fraction of ionized ClU base pairs, fins was plotted versus the percentage of ionized base pairs at a given pH, as shown in Figure 7, where the percentage of ionized base pairs is given by the following equation:

%ClU- ) 100 × [10(pH - pKa)/(1 + 10(pH - pKa))]

(2)

The pKa in eq 2 corresponds to the apparent pKa for the formation of the ionized ClU-G base pair in the active site of the polymerase. The value of the pKa was obtained by varying the pKa between 7 and 12 with each polymerase independently until the best fit was obtained for the plots shown in Figure 7.

Figure 5. Kinetic parameters of misincorporation as a function of solution pH corresponding to Table 2. (A) Pol β misincorporation. (B) AMV-RT misincorporation. (C) Klenow misincorporation. T:dGTP (1), ClU:dGTP (9).

The values obtained were 8.9, 8.5, and 8.7 for pol β, AMVRT, and Klenow, respectively. The linear plot in Figure 7, relating fins to the percentage of ionized base pairs for pol β, has a slope 1.89 × 10-4 with an intercept of 4.89 × 10-4 and an r2 value of 0.99. For AMV-RT, the plot has a slope of 1.90 × 10-5 with an intercept of 8.50 × 10-6 and an r2 value of 0.99. For Klenow, the plot has a slope of 6.32 × 10-5 with an intercept of 6.32 × 10-4 and an r2 value of 0.99. Repair of ClU in Synthetic Oligonucleotides by Human SMUG1. The repair of ClU by the human glycosylase, hSMUG1, was examined by two separate assays. In the first

Polymerase Miscoding of 5-Chlorouracil

Chem. Res. Toxicol., Vol. 23, No. 4, 2010 745

Figure 6. Proposed base pairing schemes for ClU. (A) Neutral wobble configuration. (B) Ionized pseudo-Watson-Crick configuration.

Figure 7. Relationship between polymerase kinetic parameters and base ionization. Pol β (9): The apparent pKa is 8.9; the slope of the line is 1.89 × 10-4 with an intercept of 4.89 × 10-4; and r2 is 0.99. AMVRT (b): The apparent pKa is 8.5; the slope of the line is 1.90 × 10-5 with an intercept of 8.50 × 10-6; and r2 is 0.99. Klenow (1): The apparent pKa is 8.7; the slope of the line is 6.32 × 10-5 with an intercept of 6.32 × 10-4; and r2 is 0.99.

assay, we examined the cleavage rate constant of ClU by hSMUG1 using 32P-5′-end-labeled oligonucleotides with the same sequence and reaction conditions described previously (26). We observed that the cleavage rate of the ClU-G by hSMUG1 is at least 2 orders of magnitude faster than that of ClU-A; no cleavage of ClU-A was observed where the limit of detection of the method was 1 × 10-6 s-1 (data not shown). In the second assay, we examined the cleavage rate of ClU by hSMUG1 using MALDI-TOF-MS within the oligonucleotide sequence shown in Figure 2B. The thermodynamic stability and structure of this sequence containing ClU have been studied previously (39, 40). ClU mispaired with G was selectively cleaved from the duplex, generating an oligonucleotide containing an abasic site. The relative peak heights of cleaved and intact oligonucleotides indicate that 47% of ClU-G mispair-containing oligonucleotides were cleaved. In contrast, no cleavage activity was observed for the oligonucleotide duplexes containing T-G (Figure 8B) or ClU-A (Figure 8C). The theoretical mass of the abasic site-containing oligonucleotide is 3552.59 amu, and the observed mass is 3552.52 amu. The reduction in oligonucleotide mass was observed to be 128 amu, which reflects the displacement of ClU (-145 amu) by a hydroxyl group (17 amu).

Discussion Inflammation-Mediated Reactive Molecules Can Generate ClU. Multiple reactive species are generated during an inflammatory response, and these chemically reactive species are not selective in damaging pathogens (4-9). Among the multiple reaction products is ClU in DNA, as well as the corresponding nucleosides and nucleotides in the precursor pool (4-8). The purpose of this work was to investigate the capacity

Figure 8. Activity of hSMUG1 on synthetic oligonucleotides containing T-G, ClU-A, or ClU-G. MALDI spectra of oligonucleotides. (A) Mixture of T-G, ClU-A, and ClU-G oligonucleotide without enzyme reaction. (B) T-G oligonucleotide reacted with hSMUG1. (C) ClU-A oligonucleotide reacted with hSMUG1. (D) ClU-G oligonucleotide reacted with hSMUG1, generating an oligonucleotide with an abasic site. The cleaved ClU residue is too small to be seen with this method.

of CldU to be incorporated into the DNA of replicating mammalian cells. Furthermore, we wished to examine the potential pH-dependent miscoding properties of ClU and to examine its context-dependent repair by the human repair glycoyslase, SMUG1. The three polymerases examined here, pol β, AMV-RT, and Klenow (exo-), represent commercially available model polymerases. The polymerases examined here, as well as the oligonucleotide templates utilized here, are intended to represent model systems and are not intended to be of special significance to the potential mutagenicity or toxicity of ClU and its analogues. CldU Is Incorporated into Replicating Human Cells. When CldU is placed into tissue culture medium, mammalian cells incorporate the analogue into DNA. We observe that 10 µM concentration of CldU in the media does not alter cell division kinetics; however, the mole ratio of ClU to T in the DNA is 0.89 ( 0.02 when cells are grown for two cell doublings. Previously, it has been shown that CldU is metabolized and incorporated into DNA using antibodies that bind selectively to DNA containing halogenated bases (28-32). We have confirmed and extended these findings using a mass spectral method as reported here. In the studies reported here, CldU is more similar to BrdU in acting as a T analogue. The chloro substituent of T is sufficiently large that ClU metabolites are not efficient poisons of thymidylate synthase, although a previous study suggested that the toxicity of CldU could in part be attributed to the inhibition of thymidylate synthase (32). Furthermore, the larger size of the chloro substituent, relative

746

Chem. Res. Toxicol., Vol. 23, No. 4, 2010

to the hydrogen of uracil, allows ClU to accumulate in the DNA without removal by UNG (22). The above results suggest that ClU is a good substitute for T in cell culture. However, the T-methyl group is an important recognition point for sequencespecific DNA binding proteins (49), and it is yet to be determined whether ClU can substitute for T in all sequencespecific DNA-protein interactions. When in Template DNA, ClU Codes As a T Analogue. When in an oligonucleotide template, ClU directs the incorporation of dATP. The magnitude of the Km for the incorporation of dATP opposite ClU is 1000-fold lower than that for dGTP opposite ClU measured at pH 7.5, and the kcat for incorporation of dATP is 10-fold higher than that for dGTP for each of the three polymerases examined (Table 1). These data are in accord with the cell incorporation studies described above demonstrating that CldU functions as a thymidine analogue. Efficiency of Polymerase Insertion Opposite Template ClU Is pH-Dependent. In this study, a previously described method (42) was used to approximate Vmax/Km with increasing pH. For all three polymerases, Vmax/Km for the incorporation of dATP was observed to increase and then decrease with increasing pH with an apparent pH optimum between pH 7 and 8, depending upon the polymerase. Indistinguishable behavior was seen for the incorporation of dATP opposite template T. The observed pH-dependent behavior likely reflects pH-dependent changes on the polymerase and dNTP phosphate. Although more acidic than the imino proton of T, the imino proton of ClU is an integral part of the ClU-A base pair, as seen in a recent NMR structural study (39). The misincorporation of dGTP opposite template ClU and T was also investigated by two independent methods. In the first method, the concentration dependence for dGTP was measured at three values of pH, as shown in Figures 4, S3 (Supporting Information), and S4 (Supporting Information). Although the incorporation of dGTP opposite ClU and T appears low at pH 6.5, the incorporation of dGTP opposite ClU is clearly stimulated by increasing the pH to 8.5. The observed increased misincorporation of dGTP opposite template ClU is consistent with previous data from Goodman and co-workers with FU and BrU (42) and with the formation of an ionized base pair approximating Watson-Crick geometry (40) as shown in Figure 6. In a second series of experiments, Vmax/Km was measured as a function of pH at a selected concentration of dNTP, which was less than 15% of the Km (Table 2). Changing solution pH alters both polymerase/substrate properties, as demonstrated with dATP opposite ClU, and the template miscoding property of ClU, as demonstrated with dGTP. In order to estimate the impact of pH on the base pairing of ClU, the misinsertion frequency, fins, was determined as described in eq 1. The magnitude of fins was observed to increase continuously with increasing pH, suggesting that the ionized base pair was contributing to the increased miscoding. If the ionized form of ClU does explain the increased formation of the dGTP-ClU mispair with increasing pH, a linear relationship should exist between the fraction of the ionized form of ClU and the pH-dependent fins. The fraction of the ionized form can be determined from the pH and the apparent pKa value for the ionizing species as indicated in eq 2. In this study, values for the apparent pKa were iterated between 7 and 12, and the value that provided the best fit between the percent ionized and fins was determined. The complex or apparent pKa values measured for the three polymerases are 8.9, 8.5, and 8.7 for pol β, AMV-RT, and Klenow, respectively. The pKa for CldU monomer is approximately 7.9 (37); however, for the

Kim et al.

ClU-G base pair in a duplex oligonucleotide, the apparent pKa was recently measured to be 8.7 (40). Our values appear to correlate with the previously estimated pKa value 8.6 of the BrU-G base pair for AMV-RT (42). We believe that this data is consistent with base ionization contributing substantially to the miscoding of ClU; however, our data cannot be used to eliminate the contribution of other potential forms to ClU miscoding at lower pH (50). Similar data was observed here with the three polymerases selected for study. It is possible that divergent results might be obtained with other polymerases on other templates. ClU Is Repaired When Mispaired with G Much Faster than When Paired with A. Although ClU ionization could enhance miscoding relative to T, the mutagenic potential of ClU might be amplified by DNA repair glycosylases. While misincorporated uracil residues are rapidly repaired from U-A base pairs, in U-G mispairs or those from single-stranded substrates by uracil-DNA glycosylase (UNG), the larger 5-chloro substituent of ClU prevents the UNG removal of ClU (22). Previous studies have established that ClU is repaired by two other members of the uracil glycosylase family, TDG and hSMUG1; however, ClU is removed much more efficiently or exclusively when mispaired with G rather than when paired with A. ClU is removed by TDG when mispaired with G 3 to 5 orders of magnitude faster than when paired with A (25). In a previous study from this laboratory, hSMUG1 was shown to exploit reduced duplex stability when selecting pyrimidines for removal (27); however, the repair of ClU-A was not investigated in that study. In this study, we have extended the previous results to include ClU-A. We observe here that when examined in the same sequence, under the same conditions that led to the substantial removal of ClU mispaired with G, no cleavage of ClU paired with A is observed. The limit of detection with that method is 1 × 10-6 s-1, and therefore, the selectivity of hSMUG1 for ClU-G over ClU-A is at least 2 orders of magnitude in the C(ClU)G sequence. In the current study, we also examined the relative efficiency of repair of ClU when paired with A or mispaired with G in a sequence that has been investigated in both thermodynamic and structural studies (39, 40). ClU was paired with G or A in the duplex oligonucleotide sequence shown in Figure 2B. Upon incubation with hSMUG1, cleavage of ClU can be measured directly by MALDI-TOF analysis as shown in Figure 8. The removal of ClU results in the formation of an abasic-site containing oligonucleotide with a mass reduced by 128 amu. hSMUG1 cleaves the ClU-G duplex, as shown in Figure 8D. In contrast, no cleavage of the ClU-A duplex (Figure 8C) is observed. The free energy difference between the ClU-A and ClU-G duplexes examined in this repair assay is 2.7 kcal/mol (40). Upon the basis of the parameters described previously for hSMUG1 (27), a free energy difference of 2.7 kcal/mol would predict a discrimination between ClU-G and ClU-A of approximately 30. Although we do not wish to overinterpret the MALDI-TOF data presented here, the data presented in Figure 8 indicates that ClU-G is cleaved by hSMUG1 in the G(ClU)G sequence at a rate at least 30 times that of ClU-A. ClU-G Is Repaired Much Faster than T-G by Both TDG and hSMUG1. The primary physiological role of TDG is believed to be for removing T residues mispaired with G that arise from the deamination of 5mC. While TDG does remove T mispaired with G, it removes ClU mispaired with G between 2 and 3 orders of magnitude faster, depending upon the sequence context (25). The primary characteristic that allows discrimination between T-G and ClU-G by TDG is likely due

Polymerase Miscoding of 5-Chlorouracil

Chem. Res. Toxicol., Vol. 23, No. 4, 2010 747

mediated repair. In contrast, if the ClU-G mispair were first encountered by TDG or hSMUG1, the ClU residues would be removed, allowing repair synthesis on the incorrect G-containing template, generating a transition mutation. Because of the large number of potential damage products derived from inflammation-mediated reactive chemical species, a multitude of transitions, transversions, insertions, and deletions might result from inflammation-mediated DNA damage, and therefore, the T-A to C-G mutation resulting from ClU pairing with G might not be apparent. However, the mutagenic potential of ClU formation should be considered in future studies of inflammation-mediated mutation.

Figure 9. Repair schemes for T and ClU mispaired with guanine. The T-G and ClU-G mispairs can be repaired by either the mismatch repair (MMR) or the base-excision repair (BER) pathways. ClU is rapidly repaired by BER, potentially promoting a transition mutation. The asterisk (*) denotes the gap in the opposing strand generated as a repair intermediate.

to the differences in the electronic inductive properties of a methyl group and a chlorine substituent as seen for E. coli mismatch uracil-DNA glycosylase (22). The discrimination of hSMUG1 for ClU-G versus T-G is approximately 2 orders of magnitude and derives primarily from differences in the free energy of solvation of the chlorine substituent versus the T methyl group (27). Mutagenic Potential of ClU. ClU residues in DNA and ClU metabolites can be formed at sites of inflammation (4-8). Once formed, CldU in the precursor pool can be converted to CldUTP and incorporated into DNA (29-31). When in DNA, the ClU-A base pair is thermodynamically stable and is not subject to glycosylase removal by the predominant glycosylases found in mammalian cells (24, 25). The data reported here on in Vitro polymerase incorporation, the lack of absence of repair of ClU when paired with A, and the growth of cells in the presence of CldU with substantial incorporation into DNA are consistent with previous observations on the incorporation of CldU into cellular DNA and suggest that ClU could, therefore, be a persistent lesion in DNA. The amount of ClU in DNA would be expected to increase with increased cellular exposure to inflammation. Occasionally, the ClU residue could miscode with G. The increased miscoding potential of ClU relative to T can be attributed in part to an increased tendency to ionize at physiological pH, resulting from the electron-withdrawing property of the chloro substituent. Although the miscoding potential of ClU might be modest at physiological pH, mechanisms for repair could substantially amplify the mutagenic potential of ClU. Polymerase directed misinsertion of dGTP opposite template T could potentially be repaired by the mismatch repair (MMR) pathway. The MMR pathway recognizes structural perturbations associated with mispairs and preferentially removes the mispaired base in the newly replicated strand (51). A ClU-G mispair could potentially be repaired by MMR, although this has not yet been demonstrated. The primary pathway for the repair of a G-T mispair, particularly outside a CpG dinucleotide, would be MMR. In contrast, the ClU-G mispair is also recognized by members of the BER pathway and, in particular, hSMUG1 and TDG. In this context, MMR and BER could compete with distinctly different biological outcomes (Figure 9). If identified first by MMR, the G of a ClU-G mispair generated by polymerase misincorporation would be removed, allowing for template-

Acknowledgment. This work was supported by grants GM41336 and CA084487 from the National Institutes of Health. We thank Aurelia Espinoza for assisting in the preparation of this manuscript. Supporting Information Available: Standard Curve of CldU/T molar ratio by GC/MS analysis (Figure S1); Kinetic parameters of correct base incorporation of dATP against a template T or ClU residue as a function of solution pH (Figure S2); Gel electrophoretic analysis of the incorporation by AMVRT of dGTP against template ClU and T residues as a function of solution pH (Figure S3); Gel electrophoretic analysis of the incorporation by Klenow of dGTP against template ClU and T residues as a function of solution pH (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Srikrishna, G., and Freeze, H. H. (2009) Endogenous damageassociated molecular pattern molecules at the crossroads of inflammation and cancer. Neoplasia 11, 615–628. (2) DeNardo, D. G., and Coussens, L. M. (2007) Inflammation and breast cancer. Balancing immune response: crosstalk between adaptive and innate immune cells during breast cancer progression. Breast Cancer Res. 9, 212–221. (3) Valinluck, V., and Sowers, L. C. (2007) Inflammation-mediated cytosine damage: A mechanistic link between inflammation and the epigenetic alterations in human cancers. Cancer Res. 67, 5583–5586. (4) Dale, D. C., Boxer, L., and Liles, W. C. (2008) The phagocytes: neutrophils and monocytes. Blood 112, 935–945. (5) Henderson, J. P., Byun, J., Takeshita, J., and Heineke, J. W. (2003) Phagocytes produce 5-chlorouracil and 5-bromouracil, two mutagenic products of myeloperoxidase, in human inflammatory tissue. J. Biol. Chem. 278, 23522–23528. (6) Jiang, Q., Blount, B. C., and Ames, B. N. (2003) 5-Chlorouracil, a marker of DNA damage from hypochlorous acid during inflammation. J. Biol. Chem. 278, 32834–32840. (7) Takeshita, J., Byun, J., Nhan, T. Q., Pritchard, D. K., Pennathur, S., Schwartz, S. M., Chait, A., and Heineke, J. W. (2006) Myeloperoxidase generates 5-chlorouracil in human atherosclerotic tissue. J. Biol. Chem. 281, 3096–3104. (8) Knaapen, A. M., Gu¨ngo¨r, N., Schins, R. P., Borm, P. J., and Van Schooten, F. J. (2006) Neutrophils and respiratory tract DNA damage and mutagenesis: a review. Mutagenesis 21, 225–236. (9) Kang, J. I., and Sowers, L. C. (2008) Examination of hypochlorous acid-induced damage to cytosine residues in a CpG dinucleotide in DNA. Chem. Res. Toxicol. 21, 1211–1218. (10) Litman, R. M., and Pardee, A. B. (1960) The induction of mutants of bacteriophage T2 by 5-bromouracil. III. Nutritional and structural evidence regarding mutagenic action. Biochim. Biophys. Acta 42, 117– 130. (11) Sternglanz, H., and Bugg, C. E. (1975) Relationship between the mutagenic and base-stacking properties of halogenated uracil derivatives. The crystal structure of 5-chloro- and 5-bromouracil. Biochim. Biophys. Acta 378, 1–11. (12) Noskov, V., Negishi, K., Ono, A., Matsuda, A., Ono, B., and Hayatsu, H. (1994) Mutagenicity of 5-bromouracil and N6-hydroxyadenine studied by yeast oligonucleotide transformation assay. Mutat. Res. 308, 43–51. (13) Sowers, L. C., Eritja, R., Kaplan, B. E., Goodman, M. F., and Fazakerley, G. V. (1987) Structural and dynamic properties of a

748

(14) (15)

(16)

(17)

(18)

(19) (20) (21) (22) (23) (24)

(25)

(26)

(27)

(28) (29)

(30)

(31)

Chem. Res. Toxicol., Vol. 23, No. 4, 2010 fluorouracil-adenine base pair in DNA studied by proton NMR. J. Biol. Chem. 262, 15436–15442. Kremer, A. B., Mikita, T., and Beardsley, G. P. (1987) Chemical consequences of the incorporation of 5-fluorouracil into DNA as studied by NMR. Biochemistry 26, 391–397. Fazakerley, G. V., Sowers, L. C., Eritja, R., Kaplan, B. E., and Goodman, M. F. (1987) Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR. J. Biomol. Struct. Dyn. 5, 639–650. Sowers, L. C., Eritja, R., Kaplan, B., Goodman, M. F., and Fazakerley, G. V. (1988) Equilibrium between a wobble and ionized base pair formed between fluorouracil and guanine in DNA as studied by proton and fluorine NMR. J. Biol. Chem. 263, 14794–14801. Sowers, L. C., Goodman, M. F., Eritja, R., Kaplan, B., and Fazakerley, G. V. (1989) Ionized and wobble base-pairing for bromouracil-guanine equilibrium under physiological conditions. A nuclear magnetic resonance study on an oligonucleotide containing a bromouracilguanine base-pair as a function of pH. J. Mol. Biol. 205, 437–447. Patel, D. J., Kozlowski, S. A., Marky, L. A., Rice, J. A., Broka, C., Dallas, J., Itakura, K., and Breslauer, K. J. (1982) Structure, dynamics and energetics of deoxyguanosine·thymidine wobble base pair formation in the self-complementary d(CGTGAATTCGCG) duplex in solution. Biochemistry 21, 437–444. Parker, W. B., and Cheng, Y. C. (1990) Metabolism and mechanism of action of 5-fluorouracil. Pharmacol. Ther. 48, 381–395. Wyatt, M. D., and Wilson, D. M., III. (2009) Participation of DNA repair in the response to 5-fluorouracil. Cell. Mol. Life Sci. 66, 788– 799. Wibley, J. E. A., Waters, T. R., Haushalter, K., Verdine, G. L., and Pearl, L. H. (2003) Structure and specificity of the vertebrate antimutator uracil-DNA glycosylase SMUG1. Mol. Cell 11, 1647–1659. Liu, P., Burdzy, A., and Sowers, L. C. (2002) Substrate recognition by a family of uracil-DNA glycosylases: UNG, MUG and TDG. Chem. Res. Toxicol. 15, 1001–1009. Liu, P., Burdzy, A., and Sowers, L. C. (2003) Repair of the mutagenic DNA oxidation product, 5-formyluracil. DNA Repair 2, 199–210. Bennett, M. T., Rodgers, M. T., Hebert, A. S., Ruslander, L. E., Eisele, L., and Drohat, A. C. (2006) Specificity of human thymine DNA glycosylase depends on N-glycosidic bond stability. J. Am. Chem. Soc. 128, 12510–12519. Morgan, M. T., Bennett, M. T., and Drohat, A. C. (2007) Excision of 5-halogenated uracils by human thymine DNA glycosylase. Robust activity for DNA contexts other than CpG. J. Biol. Chem. 282, 27578– 27586. Liu, P., Theruvathu, J. A., Darwanto, A., Lao, V. V., Pascal, T., Goddard, W. A., and Sowers, L. C. (2008) Mechanisms of base selection by the Escherichia coli mispaired uracil glycosylase. J. Biol. Chem. 283, 8829–8836. Darwanto, A., Theruvathu, J. A., Sowers, J. L., Rogstad, D. K., Pascal, T., Goddard, W. A., III., and Sowers, L. C. (2009) Mechanism of base selection by human single-stranded selective monofunctional uracil-DNA-glycosylase. J. Biol. Chem. 284, 15835–15846. Visser, D. W., Frisch, D. M., and Huang, B. (1960) Synthesis of 5-chlorodeoxyuridine and a comparative study of 5-halodeoxyuridines in E. coli. Biochem. Pharmacol. 5, 157–164. Jaunin, F., Visser, A. E., Cmarko, D., Aten, J. A., and Fakan, S. (1998) A new immunocytochemical technique for ultrastructural analysis of DNA replication in proliferating cells after application of two halogenated deoxyuridines. J. Histochem. Cytochem. 46, 1203–1209. Svetlova, M., Solovjeva, L., Blasius, M. M., Shevelev, I., Hubscher, U. U., Hanawalt, P., and Tomilin, N. (2005) Differential incorporation of halogenated deoxyuridines during UV-induced DNA repair synthesis in human cells. DNA Repair 4, 359–366. Yamada, K., Semba, R., Ding, X., Ma, N., and Nagahama, M. (2005) Discrimination of cell nuclei in early S-phase, mid-to-late S-phase, and G2/M-phase by sequential administration of 5-bromo-2′-deoxyuridine and 5-chloro-2′-deoxyuridine. J. Histochem. Cytochem. 53, 1365–1370.

Kim et al. (32) Brandon, M. L., Mi, L.-J., Chaung, W., Teebor, G., and Boorstein, R. J. (2000) 5-chloro-2′-deoxyuridine cytotoxicity results from base excision repair of uracil subsequent to thymidylate synthase inhibition. Mutat. Res. 459, 161–169. (33) Heartlein, M. W., O’Neill, J. P., Pal, B. C., and Preston, R. J. (1982) The induction of specific-locus mutations and sister-chromatid exchanges by 5-bromo and 5-chloro-deoxyuridine. Mutat. Res. 92, 411– 416. (34) DuFrain, R. J. (1984) Probing sister chromatid exchange formation with halogenated pyrimidines. Basic Life Sci. 29, 41–58. (35) Zwanenburg, T. S., Hansson, K., Darroudi, F., Van Zeeland, A. A., and Natarajan, A. T. (1985) Effects of 3-aminobenzamide on Chinese hamster cells treated with thymidine analogues and DNA-damaging agents. Chromosomal aberrations, mutations and cell-cycle progression. Mutat. Res. 151, 251–262. (36) Michishita, E., Kurahashi, T., Suzuki, T., Fukuda, M., Fujii, M., Hirano, H., and Ayusawa, D. (2002) Changes in nuclear matrix proteins during the senescence-like phenomenon induced by 5-chlorodeoxyuridine in HeLa cells. Exp. Gerontol. 37, 885–890. (37) Privat, E. J., and Sowers, L. C. (1996) A proposed mechanism for the mutagenicity of 5-formyluracil. Mutat. Res. 354, 151–156. (38) La Francois, C. J., Jang, Y. H., Cagin, T., Goddard, W. A., III., and and Sowers, L. C. (2000) Conformation and proton configuration of pyrimidine deoxynucleoside oxidation damage products in water. Chem. Res. Toxicol. 13, 462–470. (39) Theruvathu, J. A., Kim, C. H., Rogstad, D. K., Neidigh, J. W., and Sowers, L. C. (2009) Base-pairing configuration and stability of an oligonucleotide duplex containing a 5-chlorouracil-adenine base pair. Biochemistry 48, 7539–7546. (40) Theruvathu, J. A., Kim, C. H., Darwanto, A., Neidigh, J., and Sowers, L. C. (2009) pH-dependent configurations of a 5-chlorouracil-guanine base pair. Biochemistry 48, 11312–11318. (41) Driggers, P. H., and Beattie, K. L. (1988) Effect of pH on the basemispairing properties of 5-bromouracil during DNA synthesis. Biochemistry 27, 1729–1735. (42) Yu, H., Eritja, R., Bloom, L. B., and Goodman, M. F. (1993) Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine. J. Biol. Chem. 268, 15935–15943. (43) Kumar, R., Wiebe, L. I., and Knaus, E. E. (1994) A mild and efficient methodology for the synthesis of 5-halogeno uracil nucleosides that occurs via a 5-halogeno-6-azido-5,6-dihydro intermediate. Can. J. Chem. 72, 2005–2010. (44) Nielsen, J., Taagaard, M., Marugg, J. E., van Boom, J. H., and Dahl, O. (1986) Application of 2-cyanoethyl N, N, N′, N′-tetraisopropylphosphorodiamidite for in situ preparation of deoxyribonucleoside phosphoramidites and their use in polymer-supported synthesis of oligodeoxyribonucleotides. Nucleic Acids Res. 14, 7391–7403. (45) Herring, J. L., Rogstad, D. K., and Sowers, L. C. (2009) Enzymatic methylation of DNA in cultured human cells studied by stable isotope incorporation and mass spectrometry. Chem. Res. Toxicol. 22, 1060– 1068. (46) Randall, S. K., Eritja, R., Kaplan, B. E., Petruska, J., and Goodman, M. F. (1987) Nucleotide insertion kinetics opposite abasic lesions in DNA. J. Biol. Chem. 262, 6864–6870. (47) Boosalis, M. S., Petruska, J., and Goodman, M. F. (1987) DNA polymerase insertion fidelity. J. Biol. Chem. 262, 14689–14696. (48) Darwanto, A., Farrel, A., Rogstad, D. K., and Sowers, L. C. (2009) Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Biochem. 394, 13–23. (49) lvarie, R. (1987) Thymine methyls and DNA-protein interactions. Nucleic Acids Res. 15, 9975–9983. (50) Petruska, J., Goodman, M. F., Boosalis, M. S., Sowers, L. C., Cheong, C., and Tinoco, I., Jr. (1988) Comparison between DNA melting thermodynamics and DNA polymerase fidelity. Proc. Natl. Acad. Sci. U.S.A. 85, 6252–6256. (51) Modrich, P. (2006) Mechanisms in eukaryotic mismatch repair. J. Biol. Chem. 281, 30305–30309.

TX900302J