From the Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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A major stable oxidation product of DNA cytosine
is uracil glycol (Ug). Because of the potential of Ug to be a strong
premutagenic lesion, it is important to assess whether it is a blocking
lesion to DNA polymerase as is its structural counterpart, thymine
glycol (Tg), and to evaluate its pairing properties. Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA
polymerase I Klenow fragment (exo). During translesion
DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence
contexts examined. Furthermore, only dAMP was incorporated opposite
template Ug and Tg and the kinetic parameters of incorporation showed
that dAMP was inserted opposite Ug more efficiently than opposite Tg. Ug opposite G and A was also recognized and removed in
vitro by the E. coli DNA repair glycosylases,
endonuclease III (endo III), endonuclease VIII (endo VIII), and
formamidopyrimidine DNA glycosylase. The steady state kinetic
parameters indicated that Ug was a better substrate for endo III and
formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII,
however, Tg was a better substrate.
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INTRODUCTION |
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Free radical-induced DNA damage is a substantial contributor to
the spontaneous mutational burden and has been implicated in a variety
of disease processes including cancer. Accumulation of free
radical-induced DNA damage has also been associated with aging (1-3).
A significant number of free radical-induced mutations produced by
oxidizing agents and ionizing radiation are C T transitions (4-8),
thus oxidized cytosine residues appear to play an important role in
oxidative mutagenesis. Hydroxyl radicals, the principal damaging
species produced by oxidizing agents, interact with cytosine residues
principally by addition to the 5,6-double bond (9, 10). A major
oxidation product of DNA cytosine is cytosine glycol which is unstable
and deaminates to uracil glycol (Ug)1 (11). Cytosine glycol
can also dehydrate to form 5-hydroxycytosine (5-OHC) (11-13), while
5-hydroxyuracil (5-OHU) arises from sequential deamination and
dehydration (11). Depending on the oxidizing agent used, uracil glycol
and 5-hydroxycytosine are formed at comparable levels in DNA and
furthermore, the background level of these lesions is high in DNA
extracted from untreated cells (11).
Oxidized pyrimidine lesions are repaired by a highly conserved process
called base excision repair (for reviews, see Refs. 14 and 15). The
damaged pyrimidines are recognized by a class of enzymes called DNA
glycosylases. The principal activities in Escherichia coli
that recognize oxidized pyrimidines are endonuclease III (endo III) and
endonuclease VIII (endo VIII) (see Refs. 14 and 15, and references
therein); formamidopyrimidine DNA glycosylase (Fpg) has also been shown
to recognize oxidized pyrimidines in vitro (16). Upon
recognition of an oxidized pyrimidine by a DNA glycosylase, the
N-glycosylic bond is cleaved releasing the free base. This
is followed by cleavage of the phosphodiester backbone by an associated
DNA lyase activity which leaves a blocked 3' terminus in the resulting
nick (Refs. 14 and 15 and references therein). The block, either an
,
-unsaturated aldehyde or a phosphate must be removed by the
phosphodiesterase or phosphatase activity of another class of enzymes,
the 5' AP endonucleases (Refs. 14 and 15 and references therein). This
results in a single base gap which is filled in by DNA polymerase and
sealed by DNA ligase (Refs. 14 and 15 and references therein).
If the lesion is not repaired prior to its encounter with the replication fork, it can either block DNA polymerase and thus be potentially lethal or it can be bypassed by DNA polymerase and be potentially mutagenic depending on its ability to mispair. Of the modified pyrimidine lesions retaining the intact pyrimidine ring that have been studied to date, only thymine glycol (Tg) is a strong blocking lesion to DNA polymerases in vitro (17-20) and is lethal in vivo (21-23). Other free radical-induced pyrimidine lesions, such as dihydrothymine (24), 5-OHU (25), and 5-OHC (25), do not block DNA polymerases and are readily bypassed. Dihydrothymine always pairs with A and is not a mutagenic lesion (26). 5-OHC can pair with A in vitro (25) and has been shown to be mutagenic in E. coli (27). 5-OHU always pairs with A in vitro (25) and because it is derived from C, it is a potentially important premutagenic lesion.
Oxidized deoxynucleoside triphosphates from the nucleotide pools can
also be incorporated into DNA; these may pair correctly or mispair.
5-OHdCTP and 5-OHdUTP are efficient substrates for E. coli
DNA polymerase I Klenow fragment (Kf) (28); 5-OHdUTP is incorporated in
place of A and thus, if incorporated from the nucleotide pool, would
not be mutagenic. 5-OHdCTP can be misincorporated as T and thus could
be mutagenic. 8-Oxo-dGTP and 8-oxo-dATP can also be incorporated into
DNA; 8-oxo-dGTP is often misincorporated as T leading to T G
transversions (29, 30). We have previously reported the synthesis of
dUgTP and shown it to be a reasonably efficient substrate for DNA
polymerase I Kf (31). This is in contrast to its structural relative,
dTgTP, which is a poor substrate (24, 31). Both dUgTP and dTgTP are
incorporated in place of T (31) and thus would not be potentially
mutagenic if incorporated from oxidized nucleotide pools.
Here we report the enzymatic processing of Ug contained in template DNA
and show that Ug is readily bypassed by the model enzyme, E. coli DNA polymerase I Kf(exo), in contrast to Tg
which is poorly bypassed except in certain sequence contexts. Only dAMP can be incorporated into DNA opposite Ug. Finally, Ug in DNA is a good
substrate for endonucleases III and VIII and to a lesser extent for
Fpg, but it cannot be removed by uracil DNA glycosylase.
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EXPERIMENTAL PROCEDURES |
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Chemicals, Enzymes, and DNA--
dTgTP and dUgTP were prepared
as described previously (24, 31); the 2'-deoxynucleoside triphosphates
used in the DNA polymerase reactions and the Mono-Q 5/5 column were
purchased from Pharmacia; Partisphere SAX, 0.4 × 12.5 cm, column
was obtained from Whatman; 2',3'-dideoxynucleoside triphosphates,
Klenow fragment (10 µM/µl), Sequenase version 2.0 (13 µM/µl), T4 DNA ligase (5 µM/µl), and shrimp alkaline phosphatase were obtained from U. S. Biochemical Corp.; terminal transferase (10 µM/ml) and T4
polynucleotide kinase were purchased from Boehringer Mannheim; uracil
DNA glycosylase (100 µM/µl) was obtained from Epicentre
Technologies; E. coli endo III (10 µM), endo
VIII (200 µM), and Fpg DNA glycosylase (6.6 µM) were purified as described previously (16).
[-32P]ATP (>5000 Ci/mmol, 10 mCi/ml) was purchased
from DuPont; ultrapure Sequagel sequencing system was obtained from
National Diagnostics.
Oligodeoxyribonucleotides--
All oligonucleotides were
obtained from Operon Technologies or synthesized by the standard
phosphoramidite method on a ABI 380A DNA synthesizer (Vermont Cancer
Center, University of Vermont). 8-Oxo-2'-deoxyguanosine
phosphoramidite, used to prepare 8-oxo-G-containing oligonucleotides,
and tetrahydrofuran phosphoramidite, used to prepare the AP-containing
oligonucleotides (catalog name "dSpacer"), were obtained from Glen
Research (Sterling, VA). Lesion-containing oligonucleotides were
deprotected in the presence of 0.25 M -mercaptoethanol following the procedure recommended by Glen Research.
Synthesis of Oligonucleotides Containing an Internal Ug or Tg-- 18-, 37-, and 45-mer oligonucleotides containing a single, internal Ug or Tg (templates 1, 2, 3, 4, and 5, Fig. 1) were prepared by a modification of a method previously described (32) using terminal deoxynucleotidyl transferase. 1-2.5 nmol of GCAGCCAAAACGTCC, CCTTCG, or CCTTCGT were incubated for 30 min at 30 °C in 65 µl of buffer containing 100 mM sodium cacodylate, pH 7.0, 1 mM CoCl2, 0.1 mM EDTA, 50 µg/ml bovine serum albumin, 0.1 mM dithiothreitol, 0.1-0.12 mM dUgTP, or 0.18-0.2 mM dTgTP and 100 units of terminal deoxynucleotidyl transferase. The oligonucleotides extended from the 3'-end with a single dUgTP or dTgTP were then high performance liquid chromatography purified on a Partisphere SAX column (0.4 × 12.5 cm, Whatman) using a linear gradient of sodium phosphate buffer, pH 6.3 (from 5 mM to 0.5 M over 60 min), containing 25% acetonitrile. The purified extended oligonucleotides, GCAGCCAAAACGTCCX, CCTTCGX, or CCTTCGTX (X = Ug or Tg), were desalted using NAP-5 columns (Pharmacia) and ligated using T4 DNA ligase with 32pGGATGGTCTGTCCCTTGAATCGATAGGGG, 32pTACTTTCCTCT, or 32pACTTTCCTCT, respectively, using the appropriate "scaffolding" oligonucleotides GACAGACCATCCAGGACGTTTTGGCTGC or AGAGGAAAGTAACGAAGG. 37-Mers (templates 4 and 5, Fig. 1) were obtained by the ligation of CCTTCGX or CCTTCGTX (X = Ug or Tg) with 32pTACTTTCCTCTTCCCTTGAATCGATAGGGG or 32pACTTTCCTCTTCCCTTGAATCGATAGGGG, respectively, using AGAGGAAAGTAACGAAGG as a scaffold. All 5'-phosphorylated oligonucleotides used for ligation contained a low specific activity 5'-32P label (about 10 nCi) for detection of the ligation products. In later experiments, the radioactivity of the internal 32P label was below the level of detection by autoradiography during the exposure time used for the detection of 5'-terminal 32P label. Ligation reactions were performed for 10-15 h at 15 °C in 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 0.1 mM EDTA, 10 mM dithiothreitol, 0.3 mM ATP, 10 µM nicked duplex oligonucleotide, and 0.5 unit/µl reaction mixture of T4 DNA ligase. After ligation, the strands containing the lesion were separated from the scaffolding oligonucleotide by electrophoresis on 12 or 20% polyacrylamide gels under denaturing conditions, electroeluted from the gel, and desalted using NAP-5 columns. The ligation efficiency of oligonucleotides containing 3'-dUg or -dTg was lower than those containing 3'-dT. The yield of ligation products of oligonucleotides without lesions was 90-95%. Under the same reaction conditions, the conversion of 3' modified oligonucleotides into ligation products ranged between 45 and 60%.
Preparation of DNA Templates and Double-stranded Oligonucleotides Containing Abasic Sites-- Appropriate amounts of templates 1, 4, and 5 were annealed to a 32P-labeled primer 1 (Fig. 1) in Buffer P (15 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 30 mM NaCl, 4 mM dithiothreitol) and treated with an excess of uracil DNA glycosylase following the manufacturer's instructions.
A double-stranded 18-mer oligonucleotide duplex containing an abasic site, which was used as a substrate for DNA repair endonucleases, was prepared by annealing 5'-32P-labeled template 2 with U in position 7 to 18-mer 2 (Fig. 1) in 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA buffer followed by treatment of the duplex with excess uracil DNA glycosylase following the manufacturer's instructions.Bypass Assay--
The bypass of Ug and Tg in different sequence
contexts was studied using various concentrations of
Kf(exo) (10
5-10
2
µM/µl). In the typical bypass assay, a primer
oligonucleotide containing the 5'-32P label was annealed to
a template with single Ug or Tg. The primer-template complex (50 nM) was incubated at 37 °C for 15 min with
Kf(exo
) in 6 µl of the reaction mixture, containing
Buffer P and four dNTP's (64 µM each). The same
conditions were used for the "running start" primer extension
reaction. Here the concentration of Kf(exo
) was 1.6 × 10
3 µM/µl and the concentration of
each of four dNTP's was 50 µM.
One Nucleotide Extension Assay--
Extension of the primer with
only one nucleotide was performed similarly to the bypass assay using 5 µM of only one of the four dNTP's in each experiment and
2.5-6 × 104 µM/ml
Kf(exo
). The reaction mixtures were incubated for 15 min
at 15 °C.
Dideoxy Sequencing Reactions-- Sequencing reactions were performed using T7 DNA polymerase (Sequenase version 2.0) following the supplier's recommendations (U. S. Biochemical Corp.).
Kinetics of Incorporation of dAMP Opposite Tg or Ug by
Kf(exo)--
To determine the kinetic parameters for
lesion bypass, a steady state kinetic assay (33) was used. The reaction
mixture was prepared by adding 3.5 µl of a solution containing primer 32pTCAAGGGACAGACCAT, annealed to (template 1, Fig. 1)
Kf(exo
) and the buffer to 2.5 µl of water solution
containing dCTP and dATP. The final mixture (6 µl) contained 0.02 unit of Kf(exo
), Buffer P, 50 nM
primer-template complex, 50 µM dCTP, and various concentrations of dATP (0.05-10 µM). Reactions were
incubated at 4 °C. Reaction times varied between 0.5 and 2 min.
Repair Enzyme Assays-- For endo III, endo VIII, and Fpg DNA glycosylase assays, the standard reaction mixture (10 µl) contained 100 nM double-stranded oligonucleotide substrate, 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, and 1-100 nM enzyme. In all experiments, the strand containing the lesion was 5'-32P-labeled. Each enzyme reaction was incubated at 37 °C for 20 min. The reactions were terminated by adding 10 µl of loading buffer and reaction products were analyzed in a 13% polyacrylamide denaturing gel. To determine kinetic parameters, a 5'-32P-labeled template 2 (Fig. 1) containing Ug or Tg at position 7 was annealed to 18-mer 2 (Fig. 1) in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA buffer. As a control for the Fpg DNA glycosylase reaction, a 24-mer 32pGAACTAGTGG(8-oxo-G)TCCCCCGGGCTGC was annealed to the appropriate 24-mer complement strand. For all substrates, the concentration range used was 20-300 nM. Endo III, endo VIII, and Fpg DNA glycosylase were used in the 2-5 nM concentration range. Aliquots of the reaction mixtures were withdrawn at 0.5-min intervals and quenched with gel loading buffer. For each substrate and enzyme, the data obtained were then fitted to calculate the apparent Km and Vmax values using the Macintosh program, k.cat (BioMetallics, Inc.).
Electrophoresis-- All DNA polymerase reactions were terminated by the addition of an equal volume of loading buffer (95% formamide, 0.05% bromphenol blue, 0.05% xylene cyanol, and 20 mM EDTA). Reaction products were analyzed by electrophoresis on 0.4 mm, 13% polyacrylamide gels containing 8 M urea. The gels were electrophoresed in 50 mM Tris borate, 2 mM EDTA buffer, pH 8.3, for 1.5-3 h at 2000 V, dried under vacuum, and exposed to x-ray film. The radioactivity in the bands corresponding to the products of enzymatic reactions were analyzed using a Model GS-250 Molecular Imager System (Bio-Rad).
Computational Analysis-- Tg is assumed to be the A conformer (as defined by Miller and Miaskiewicz (34)), the 5R,6S-stereoisomer in which the hydroxyl groups at the C5 and C6 positions are in the pseudoequatorial (5-eq) and pseudoaxial (6-ax) positions, respectively (see Kung and Bolton (35) and references therein for a discussion of the identification of the cis-stereoisomer of Tg formed in DNA, and Miaskiewicz and Miller (36) for ab initio determination of the relative stabilities of the 5-eq/6-ax and 5-ax/6-eq enantiomers of Tg). The 5-eq/6-ax enantiomers of Ug were found to be the most stable cis-isomers of Ug based on ab initio calculations2 and the 5R,6S species was identified as the dominant conformer obtained from the synthesis procedure using NMR (31).
The methods for obtaining geometries and force field parameters for Tg and Ug have been described previously (31). Briefly, geometries were obtained using Gaussian 94 (Gaussian, Inc.), partial charges were obtained, based on the Gaussian 94 calculations, using the RESP procedure of Kollman and co-workers (37), and the remaining force field parameters were chosen from the AMBER set (38). Damaged bases were modeled into DNA by replacing the pyrimidine of the central base pair in 7-base pair B-DNA fragments as described previously (31), and LeaP (39) was used to add neutralizing counterions and explicit water molecules. Energy minimization and molecular dynamics of the counterions and solvent were followed by energy minimization and short molecular dynamics simulations of the entire systems. Nucleic acid structure parameters (40) were measured using the Biopolymer module of Insight® II from Biosym/MSI (which is based on the NEWHEL 91 program suite of Dickerson and co-workers). We note that the numerous limitations of the molecular modeling procedure used here (e.g. the absence of polymerase, the length of the DNA fragments, the duration of the simulations, and the use of B-DNA for the starting conformation) make it impossible to derive quantitative conclusions based on structure. The objective of the molecular mechanics calculations was to relieve the substantial steric repulsions that resulted from introduction of the damaged bases in B-DNA, and in doing so provide a plausible illustration of the impact of the difference between the A conformers of Tg and Ug on DNA structure. During the brief simulation, the Watson-Crick hydrogen bonds of the undamaged pairs remained essentially intact. ![]() |
RESULTS |
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Translesion Synthesis Past Ug and Tg--
Thymine glycol has been
shown to be a strong block to several DNA polymerases (17-20). When Tg
was randomly introduced into DNA, it constituted an absolute block to
Kf in all but one sequence context, 5'-C(Tg)Pu-3' (19, 26). To compare
the bypass of Ug and Tg by Kf(exo), three different DNA
templates containing Ug or Tg in the sequence contexts 5'-CXG-3' (known
Tg bypass context, see Refs. 19, 24, and 26), 5'-GXT-3', or
5'-TXA-3', where X was Ug or Tg (templates 1, 4, and 5, respectively, Fig. 1), were
prepared. As a "no lesion" control, templates 1, 4, and 5 contained
T in place of Tg or Ug and as a "complete blockage" control,
templates 1, 4, and 5 contained an abasic site. The same
5'-32P-labeled primer 1 was used with all three templates.
Figs. 2 and
3 show the results of translesion
synthesis using these templates with different concentrations of
Kf(exo
). With the "bypass sequence context" (Fig. 2),
both Tg and Ug were bypassed at high concentrations (10
2
and 10
3 µM/µl) of Kf(exo
)
(lanes 9, 10, and 13, 14, Fig. 2). At
10
2 µM/µl of Kf(exo
), even
an abasic site was successfully bypassed (lane 1, Fig. 2);
however, an almost complete block was observed with an abasic site
(lane 2, Fig. 2) at 10
3 µM/µl
where Tg was bypasssed (lane 10, Fig. 2). Lowering the polymerase concentration further (10
4
µM/µl) caused substantial termination at the lesion
site in the case of Tg (lane 11, Fig. 2), but much less
termination in the case of Ug (lane 15, Fig. 2). As can be
seen in Fig. 3, Tg and Ug in the sequence context 5'-TXA-3'
exhibited similar behavior (lanes 5-12, Fig. 3, panel
B) to that observed in the context 5'-CXG-3'; Tg and Ug
were bypassed at high concentrations of polymerase (lanes 5, 6, 9, and 10, Fig. 3, panel B) while Ug was
more easily bypassed than Tg at lower concentrations (compare
lane 7 with lane 11, Fig. 3, panel
B).
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Specificity of Nucleotide Incorporation Opposite Ug by
Kf(exo) DNA Polymerase--
The specificity of
Kf(exo
) catalyzed nucleotide incorporation opposite Ug or
Tg was determined from both "standing" and running start positions.
For the standing start reaction, a "one nucleotide extension" assay
(28) was used. Templates 1, 2, and 3 containing Ug or Tg were annealed
to primers 2, 8, or 9 (Fig. 1), respectively, which terminate one
nucleotide prior to the lesion. In each experiment under the standing
start format, dNTPs were added one at a time. Fig.
4 shows that in the same three sequence
contexts used for measuring bypass (5'-CXG-3',
5'-GXT-3', and 5'-TXA-3', where X was
Ug or Tg) only dAMP was incorporated opposite Ug or Tg (lane 2, all panels). The incorporation efficiency of dAMP opposite Ug and
Tg in all sequence contexts was found to be lower then opposite T in
the control experiment (Fig. 4, compare lanes 2 for
different templates). An interesting observation was made with
templates 2 and 3 where significant amounts of dGMP were misincorporated opposite T (lane 1, panel B, Fig. 4). Trace
amounts of dTMP and dCMP incorporation opposite T were also detected
(lanes 3 and 4, panel B, Fig. 4) probably due to
loop out of T with dTMP or dCMP then incorporated opposite the 5'-G.
High levels of misincorporation for this particular sequence context
were observed previously (28). This is in contrast to the running start
experiments with all 4 nucleotides (Fig.
5), where only A was observed in the
full-length product opposite T in position 7. Misincorporation of dGMP
opposite T was also found at position 8 of template 3 but to a lesser
extent (panel C, corresponding section, lane 1,
Fig. 4). However, no misincorporation opposite Ug and Tg at positions 7 or 8 of template 2 or 3 was detected. Either the presence of Ug or Tg
in the template reduced misincorporation, or more likely,
misincorporation was not detectable due to the overall lower level of
incorporation opposite Ug and Tg. For example, primer 8 was extended
completely with T in the template but only about 50% opposite Ug or Tg
in the template (compare, for example, all three lanes 2, panel
B, Fig. 4). No misincorporation opposite T was observed in the
sequence context of template 1 (panel A, Fig. 4).
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Specificity of Nucleotide Incorporation Opposite Ug from a Running
Start Position--
The specificity of nucleotide incorporation
opposite Ug was also examined in a running start reaction by the
approach that was used to determine the base pairing specificity of
8-oxo-G (41) and the 5-hydroxypyrimidines (28). This same approach with
several technical improvements was used recently to determine the
miscoding properties of
3,N4-etheno-2'-deoxycytidine (42). Fig. 5
shows the results using 5'-32P-labeled primer 10 annealed
to template 2, containing Ug or Tg or T at position 7. Primer 10 terminates three nucleotides prior to the lesion in the template
strand. Four control 5'-32P-labeled 18-mers (18-mers 1-4,
Fig. 1) with G, A, C, or T at position 12 were used to compare their
mobility in a 13% polyacrylamide gel (Fig. 5, lanes 1-4)
with the mobility of full-length products of Kf(exo)
catalyzed extension of primer 10. Fig. 5 shows that only dAMP was
incorporated opposite Ug, Tg, and T during translesion DNA synthesis.
The amounts of full-length product obtained with the Ug and Tg
templates (Fig. 5, lanes 6 and 7) were lower than
with the T template (lane 5), probably reflecting the poorer
bypass of Ug and Tg lesions in this template.
Steady State Kinetic Analysis of A Insertion Opposite Ug or
Tg--
Both standing and running start experiments showed that only
dAMP was incorporated opposite Ug and Tg by Kf(exo).
Steady state kinetic assays were used to quantify the reactions and to
compare the kinetic parameters for incorporation of dAMP opposite T, Ug
and Tg. For this purpose, a 16-mer primer (primer 3, Fig. 1) was
annealed to a 45-mer template 1 containing T, Ug or Tg at position 16 (Fig. 1) in the "bypassable context" 5'-CXG-3'. The
target site, the lesion in the template, was placed at the third
position downstream from the primer, preceded by two G residues. This
provided a way to determine the kinetic parameters for the incorporation of dAMP, opposite T, Ug, or Tg by Kf(exo
)
in the presence of dCTP and dATP from a running start as described by
Boosalis et al. (33). The primer was 32P-labeled
and the extension products were quantified after polyacrylamide gel
electrophoresis. The apparent Km and
Vmax values for insertion (Table
I) were determined based on the relative velocity of primer extension with dATP measured as
I3/I2 at
t = 1 min, where I3 and
I2 correspond to the radioactivity of the extension product at sites 3 and 2, respectively, and expressed as
percentage of total primer (33).
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Extension of Primers Paired with Ug by Klenow
(Exo)--
The relative stability of the A-Ug pair was
additionally confirmed by the extension of primers with 3' dG, dA, dT,
or dC opposite template Ug. Template 1 with the Tg "bypassable
sequence context" 5'-CXG-3' with Tg, Ug, or T in position
16 was annealed to primers 4-7 (Fig. 1). This template was then
extended by Kf(exo
) and dGTP. Fig.
6 shows that dA opposite Ug was extended
by dGTP; a trace amount of dG extension was also observed, probably due to slippage of the primer template. Little or no extension of any
primer opposite Tg was observed. Extension of both dA and dG primers
opposite T was observed; the latter again probably due to slippage.
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Recognition of Uracil Glycol by the E. coli DNA
Glycosylases--
Endonucleases III and VIII have been shown to
recognize a broad spectrum of oxidized pyrimidines in vitro
and to function in vivo to repair these lesions (for a
review, see Ref. 15). Fpg also can remove oxidized pyrimidines (16).
Fig. 9 shows the activity of endo III,
endo VIII, and Fpg on duplex substrates containing Ug opposite A and Ug
opposite G. The results show that Ug opposite G, the substrate
resulting from oxidation of cytosine in DNA, and Ug opposite A, the
substrate resulting from incorporation of dUgTP from the nucleotide
pools, were equally well cleaved by all three enzymes. Also, the
reaction products resulting from (endo III) or from
(endo
VIII and Fpg) elimination were the same for each enzyme on all three
substrates (data not shown). Steady state kinetic parameters determined
for the three enzymes with both Ug- and Tg-containing substrates (Table
II) suggest that Ug was a better
substrate for endo III and Fpg than Tg, while Tg was a better substrate
for endo VIII. For endo III and Fpg, the Ug substrate was about
2.3-fold and 4.5-fold better than the Tg substrate, respectively. In
contrast, for endo VIII, the Tg substrate was 3-fold better than the Ug
substrate. Analysis by mass spectrometry of the released product after
endo III digestion of the Ug substrate showed it to have a molecular
mass corresponding to uracil glycol (data not shown).
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Molecular Modeling of DNA Sequences Containing Ug and Tg-- Clark et al. (43) and Miaskiewicz et al. (44) interpreted the biochemical effects of the presence of Tg in DNA by constructing a molecular model of DNA containing Tg and comparing structures observed after energy minimization (43) or during molecular dynamics simulations (44) with similarly obtained structures of DNA fragments containing only undamaged bases. To provide a qualitative comparison of the effects of the presence of Tg and Ug on B-DNA, we performed limited molecular dynamics simulations (Fig. 10) with Ug and Tg in the Tg stop sequence 5'-GXT-3'. As can be seen, with the steric repulsions relieved in the molecular dynamics simulation, the methyl group in Tg causes a significant increase in the distance between the Tg and the 5'-G with respect to B-DNA while Ug (or T) does not.
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DISCUSSION |
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Uracil glycol, a major oxidative product of DNA cytosine, when
present in template DNA appears to be readily bypassed by
Kf(exo) (Figs. 2 and 3). Furthermore, only dAMP was
inserted opposite Ug by Kf(exo
) in three different
sequence contexts and in both standing and running start reactions
(Figs. 4 and 5). Kinetic analysis, with the sequence context
5'-CXT-3', showed that the insertion of dAMP opposite Ug was
only about 2.4-fold less efficient than opposite T in contrast to
insertion of dAMP opposite Tg which was about 5.9-fold less efficient
than opposite T (Table I). Primers paired with Ug were also readily
extended by Kf(exo
) (Figs. 6 and 7), suggesting that the
A-Ug pair is relatively stable.
The two major factors determining the mutability of a given lesion are its ability to be bypassed by DNA polymerases and the ability of the lesion to mispair. The prototypic mutagenic lesion that fills both criteria is uracil derived from DNA cytosine which is non-blocking to DNA polymerases and always pairs with A. Similarly, uracil glycol, which is also derived from DNA cytosine, is bypassed well, at least by E. coli DNA polymerase I, and at the same time always pairs with A, suggesting that it will be a potent premutagenic lesion. Accordingly, it seems propitious that uracil glycol be removed from oxidized DNA prior to replication. The data in Fig. 9 and Table II show that Ug is recognized and removed in vitro by the E. coli DNA glycosylases that act on oxidized DNA bases. It is a good substrate for endonuclease III, better than its benchmark lesion thymine glycol; however, Ug is less efficiently removed than thymine glycol by endonuclease VIII. Recently, others have also shown Ug to be a substrate for endo III (45). Interestingly when the Vmax/Km are compared for Fpg protein, uracil glycol is removed at about 25% the efficiency of its benchmark lesion, 8-oxoguanine. If these studies can be extrapolated to the cell, uracil glycol is clearly a substrate for all three activities. However, in contrast to another oxidized product of cytosine, 5-hydroxyuracil (16), uracil glycol is not a substrate for uracil DNA glycosylase.
Uracil glycol is a close structural relative of thymine glycol which
has been shown to be a strong block to a number of DNA polymerases
(17-20) and to be a lethal lesion in vivo (21-23). In this
study, the properties of uracil glycol were compared side by side with
those of thymine glycol. As was shown previously for randomly
introduced thymine glycol lesions and Kf (19, 24, 26), thymine glycol,
site specifically introduced into sequences that were bounded on the 5'
side by purines constituted a strong block to Kf(exo)
(Fig. 3). As was also observed with randomly introduced lesions and Kf
(19, 24, 26), when thymine glycol was bounded on the 5' side with
cytosine and on the 3' side with purines, it was more readily bypassed
by Kf(exo
) (Fig. 2). Similar results were obtained with
an osmium tetroxide-oxidized T site specifically introduced into an
oligonucleotide (46), and in vivo (47). In previous reports
(19, 24, 26), in the presence of proofreading, the predominant Tg
bypassable site was 5'-CTgA-3' with bypass at 5'-CTgG-3' also observed.
In the present studies, a 5'-T also facilitated bypass of Tg. Since
bypass of Tg (46), abasic sites (48), and acetylaminofluorene-G adducts (49) by Kf, abasic sites by T4
polymerase3 and aminofluorene
adducts by T7 polymerase (50) is greatly facilitated by eliminating 3'
5' proofreading, it might be that the sequence context specificity
of Tg bypass is relaxed in the absence of proofreading. The difference
between the Tg bypassable and non-bypassable sequences was readily
apparent at low polymerase concentrations when the polymerase was
loaded onto the primer downstream from the lesion site. Much higher
concentrations of polymerase were required to achieve bypass of thymine
glycol when the primer was annealed opposite (Fig. 8), one base before
or one base beyond (data not shown) the lesion site, although at high
polymerase concentrations, bypass from a standing start was observed at
the 5'-CTgG-3' site but not at 5'-GTgT-3' site (Fig. 8).
Taken together, the studies of Kf interactions with uracil glycol and
thymine glycol show that in all sequence contexts, uracil glycol was
more readily bypassed by Kf(exo) than thymine glycol,
suggesting that the C5 methyl group of the Tg significantly
affects the interaction between the enzyme and the template/primer.
Furthermore, in particular sequence contexts, thymine glycol was more
readily bypassed than in other sequence contexts, suggesting that the
surrounding DNA sequence influences the presentation of the lesion to
the DNA polymerase. This effect must be polymerase specific since it
has been previously shown that when Tg is present in the bypassable site for Kf, it is a strong stop site for T4 polymerase (26). Strong
sequence context effects on polymerase-DNA lesion interactions have
been previously observed with lesions other than Tg (Refs. 49 and 51,
for a review, see Ref. 52 and references therein). Sequence context
also affects formation and extension of a proper base pair
versus a mispair, and mutational spectra obtained with a
number of lesions under conditions where sequence context effects on
lesion production and repair could not account for the results show
strong sequence context effects (for a review, see Ref. 52).
The observed rates of DNA synthesis from templates containing Tg or Ug can be discussed in the context of a plausible structural model based on the work of Clark et al. (43) and Miaskiewicz et al. (44). When the A conformer of either Tg or Ug is modeled into canonical A-DNA or B-DNA, the pseudoaxial hydroxyl at C6 is directed toward the 3' end of the template. Clark et al. (43) suggested, based on modeling studies, that a hydrogen bond occurs between this hydroxyl and N7 of a 3'-purine in the template, and suggested that this would confer sequence context dependence of polymerase bypass. (Limited molecular dynamics simulations of Tg- and Ug-containing DNA in the presence of counterions and explicit solvent molecules are consistent with the formation of such a hydrogen bond.) In contrast, steric interference of the 6-ax hydroxyl group with the methyl group at C5 of a thymine 3' to the lesion would occur in canonical A-DNA or B-DNA. In short molecular dynamics simulations, this steric interference resulted in a significant negative propeller twist of the base pair 3' to the Tg and Ug in the 5'-GXT-3' context relative to that in the 5'-CXG-3' context. It seems reasonable to expect that such steric interference on the 3' side of the template would result in a reduced rate of insertion opposite the lesion because it would interfere with stacking of Tg onto the formed primer-template helix. This is consistent with the results shown in Figs. 2 and 3, that is, the amount of product that is one base short of the lesion is greater in the 5'-GXT-3' context (Fig. 3A) for both Tg and Ug than in the two contexts containing a purine 3' to the lesion (Figs. 2 and 3B).
The significant structural difference between the A conformers of Tg and Ug is the pseudoaxial methyl group in Tg, which would be directed toward the 5' end of the template in a Watson-Crick base pair. Modeling studies (Fig. 10 and Ref. 43 and 44) suggest that this methyl group in Tg interferes with the structure of a base pair 5' to the lesion. In particular, Clark et al. (43) inferred a large change in the tilt of the base pair 5' to the lesion, and Miaskiewicz et al. (44) found a large increase in the rise associated with the 5'-ATg base pair step compared with that of undamaged DNA, as well as a more negative inclination of the base pair 5' to Tg and unstable hydrogen bonding between bases in the pair 5' to Tg. As could be expected, our simulation results indicate that the pseudoaxial hydrogen at C5 in Ug does not interfere with the base pair on the 5' side to the extent that a methyl group does. It is reasonable to expect that the presence of steric interference 5' to the lesion in the template would result in a much smaller rate of extension of the base opposite Tg relative to Ug, as pointed out by Clark et al. (43). This is consistent with the results shown in Figs. 2 and 3, i.e. Ug is in general bypassed more efficiently than Tg. Determination of the high resolution structure of T7 DNA polymerase, which is structurally homologous to DNA polymerase I, demonstrated that the incoming nucleoside triphosphate and the coding base in the template are tightly sandwiched between the primer-template complex and the O helix of the polymerase (53). It is interesting to note that the efficiency of insertion opposite Tg is reduced only 6-fold in the 5'-CTgG-3' sequence context (Table I), suggesting that there is sufficient space/flexibility to accommodate the pseudoaxial methyl group of Tg. It is possible that minor differences in the environment of the base opposite the incoming nucleoside triphosphate lead to the significant differences observed between the abilities of different polymerases to bypass Tg.
In summary, the data suggest that uracil glycol is likely to be a
potent premutagenic lesion and that unlike thymine glycol, it is
readily bypassed by Kf(exo). Thus, uracil glycol joins
the other well studied free radical-induced DNA base lesions such as
8-oxo-G, 8-oxo-A, 5-OHC, 5-OHU, and dihydrothymine as bypassable
lesions. In this regard, with the exception of the ring open and
fragmentation products, sites of base loss and possibly formamidopyrimidines, most of the well studied oxidative base lesions
are not lethal but bypassable and potentially mutagenic. Thymine glycol
appears to be a anomalous in this respect.
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ACKNOWLEDGEMENTS |
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The computational analysis was done in the Vermont Cancer Center's Molecular Modeling Facility. We are grateful to Dr. James Bigelow who performed the mass spectrometry in the Vermont Cancer Center's Bioanalytical Facility and to Dr. Agnes Dereski-Kovacs for performing the Gaussion 94 and RESP calculations.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R37 CA33657 and CA52040 from the National Cancer Institute. The Vermont Cancer Center's Core Facilities was supported by Grant CA22435 from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present Address: Pentose Pharmaceuticals, Inc., 45 Moulton St.,
Cambridge, MA 02138.
§ To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, Burlington, VT 05405. Tel.: 802-656-2164; Fax: 802-656-8749; E-mail: swallace{at}zoo.uvm.edu.
1
The abbreviations used are: Ug,
5,6-dihydroxy-5,6-dihydrouracil; dUG,
5,6-dihydroxy-5,6-dihydrodeoxyuridine; Tg,
5,6-dihydroxy-5,6-dihydrothymine; dTg,
5,6-dihydroxy-5,6-dihydrodeoxythymidine; dUgTP,
5,6-dihydroxy-5,6-dihydrodeoxyuridine 5'-triphosphate; dTgTP,
5,6-dihydroxy-5,6-dihydrodeoxythymidine 5'-triphosphate;
Kf(exo), DNA polymerase I Klenow fragment lacking
proofreading activity; endo, endonuclease; eq, pseudoequatorial; ax,
pseudoaxial.
2 A. Derecski-Kovacs, S. S. Wallace, and J. P. Bond, unpublished data.
3 Z. Hatahet and S. S. Wallace, unpublished studies.
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REFERENCES |
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