From the Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan
Received for publication, September 20, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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5-Formyluracil (fU) is a major oxidative thymine
lesion generated by ionizing radiation and reactive oxygen species. In
the present study, we have assessed the influence of fU on DNA
replication to elucidate its genotoxic potential. Oligonucleotide
templates containing fU at defined sites were replicated in
vitro by Escherichia coli DNA polymerase I Klenow
fragment deficient in 3'-5'-exonuclease. Gel electrophoretic analysis
of the reaction products showed that fU constituted very weak
replication blocks to DNA synthesis, suggesting a weak to negligible
cytotoxic effect of this lesion. However, primer extension assays with
a single dNTP revealed that fU directed incorporation of not only
correct dAMP but also incorrect dGMP, although much less efficiently.
No incorporation of dCMP and dTMP was observed. When fU was substituted
for T in templates, the incorporation efficiency of dAMP
(fA = Vmax/Km) decreased to
1/4 to 1/2, depending on the nearest neighbor base pair, and that of dGMP (fG) increased 1.1-5.6-fold.
Thus, the increase in the replication error frequency
(fG/fA for fU
versus T) was 3.1-14.3-fold. The misincorporation rate of
dGMP opposite fU (pKa = 8.6) but not T
(pKa = 10.0) increased with pH (7.2-8.6) of the
reaction mixture, indicating the participation of the ionized (or
enolate) form of fU in the mispairing with G. The resulting mismatched
fU:G primer terminus was more efficiently extended than the T:G
terminus (8.2-11.3-fold). These results show that when T is oxidized
to fU in DNA, fU promotes both misincorporation of dGMP at this site
and subsequent elongation of the mismatched primer, hence potentially mutagenic.
Faithful replication of DNA is essential for maintaining genetic
integrity of living organisms. High fidelity of DNA replication is
achieved by two cellular functions that involve discrimination of
correct versus incorrect nucleotides by DNA polymerases (1, 2) and postreplication mismatch repair (3). The overall error frequency
of DNA replication is one in 108 to 1010 base
pairs when they function properly. Fidelity of DNA replication also
relies on the structural integrity of DNA itself that serves as a
template for the newly synthesized strand. A number of endogenous and
exogenous agents have been identified to induce structural deterioration of DNA (4). Among them, reactive oxygen species generate
a very complicated spectrum of DNA damage (5, 6). These lesions are
mostly restored by the base excision repair pathway both in prokaryotic
and eukaryotic cells, but if left unrepaired, they arrest DNA synthesis
or direct misincorporation of nucleotides during DNA replication, hence
exerting deleterious effects on cells (7, 8). Replication blocks and
nucleotide misincorporation have been related to lethality and mutation
of cells, respectively, until recently. However, this concept is now
challenged by the discovery of numerous error-prone and error-free DNA
polymerases that can bypass the blocking lesions (9).
Although the past several years have witnessed the discovery of novel
lesion replicating DNA polymerases (mentioned above) as well as
remarkable progress in understanding the molecular basis for the
nucleotide discrimination mechanism by DNA polymerases (10, 11), the
assessment of genotoxic effects of structurally diverse oxidative DNA
damage largely relies on experimental data obtained from defined
lesions (7, 8, 12, 13). We have been studying the response of DNA
polymerases to the encountered oxidative thymine (13-17) and other
base (18-21) lesions. Oxidative thymine lesions formed by ionizing
radiation, Fenton-type reactions, and photosensitized reaction have
been best characterized among the four DNA bases and can be classified
into four subgroups depending on their structural features. The first
group includes C-5 The genotoxic potential of fU belonging to the fourth group has been
assessed in this (27, 28) and other (29, 30) laboratories. In our
previous approach, we synthesized 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) and studied its incorporation into DNA by DNA
polymerases. fdUTP efficiently substituted for dTTP and to a much less
extent for dCTP. Moreover, the pH-dependent variation of
the substitution efficiency for dCTP suggested involvement of an
ionized (or enolate) form of fU as a key intermediate responsible for
the mispairing with template G. Such a mutation mechanism involving
ionized bases (thymine and 5-bromouracil (BrU)) was originally
suggested by Lawley and Brookes (31). Later, the pH-dependent variation of the replication error frequency
due to ionization of BrU and 5-fluorouracil was experimentally
demonstrated by Yu et al. (32), providing the conceptional
basis of the previous studies (27, 28). Translesion bypass and
nucleotide incorporation at the site of template fU were also studied
(29, 30) using oligonucleotides containing the site-specific lesion
(33). Consistent with very efficient substitution of fdUTP for dTTP in
our study, DNA polymerase readily passed through the fU site in the
template. However, to our surprise, the primer extension study showed
that fU directed incorporation of dCMP as well as correct dAMP,
implying the formation of an fU:C mispair during DNA replication. The
discrepancy between the two studies concerning the base pairing
capacity of fU might have originated from several reasons. First,
although overall Watson-Crick geometry of a newly formed base pair
plays a dominant role in nucleotide selection by DNA polymerases
(1, 2), this process can also be affected by base pairing symmetry whether an X:Y base pair is formed from X
(template):Y (dNTP) or X (dNTP):Y
(template) (34-36). Second, the sequence context can affect the
selection of dNTP opposite the template lesion (7). Third, the base
ionization mechanism somehow does not hold when fU is present in
template DNA.
In view of the potential influences of base pairing asymmetry, the
sequence context, and deviations from the base ionization mechanism
mentioned above, we have prepared oligonucleotide templates containing
site-specific fU following the previously reported phosphoramidite
method (33), and we reexamined the base pairing capacity of template fU
with the four possible nearest neighbor base pairs. The results show
that fU in the template directs misincorporation of dGTP in a
pH-dependent manner, supporting our previous results obtained by the analysis of fdUTP incorporation.
Chemicals and Enzymes--
Ultra-pure dATP, dGTP, dCTP, and dTTP
(purity >99.3%) were purchased from Amersham Pharmacia Biotech.
5-Formyl-2'-deoxyuridine (fdU) was synthesized as described previously
(27). The phosphoramidite monomer of protected
5-(1,2-dihydroxyethyl)-2'-deoxyuridine was synthesized following the
reported procedure (33). [ Oligonucleotides--
Oligonucleotides comprising normal
components were synthesized by the standard phosphoramidite method and
purified by reversed phase HPLC. Oligonucleotides containing fU were
prepared following the reported procedure (33). First, oligonucleotides
containing 5-(1,2-dihydroxyethyl)uracil, a precursor of fU, were
synthesized by the standard phosphoramidite chemistry using a
phosphoramidite monomer of protected
5-(1,2-dihydroxyethyl)-2'-deoxyuridine and purified by reversed phase
HPLC. The oligonucleotides containing 5-(1,2-dihydroxyethyl)uracil was
treated by sodium periodate to convert 5-(1,2-dihydroxyethyl)uracil to
fU. After treatment, crude oligonucleotides were desalted by passing
through a Sephadex G-10 column and further purified by reversed phase
HPLC. The sequences of oligonucleotides used in the present study are
listed in Table I.
Composition Analysis of Oligonucleotides--
25T and 25F (0.2 OD) were incubated with nuclease P1 (1 unit) in a reaction buffer (60 µl) containing 21 mM sodium acetate (pH 5.3) and 1 mM ZnSO4 at 37 °C for 1 h. To this
solution, alkaline phosphatase buffer (30 µl) comprising 0.5 M Tris-HCl (pH 9.0) and 10 mM MgCl2
and alkaline phosphatase (3 units) were added, and the reaction mixture
was further incubated at 37 °C for 2 h. The sample was passed
through a molecular weight cut-off filter (Mr = 10,000), and an aliquot of the filtrate was analyzed by HPLC equipped
with a C18 WS-DNA column (4.6 × 150 mm, Wako). The sample was
eluted by a gradient of methanol in 10 mM sodium phosphate buffer (pH 7.4) at a flow rate 0.8 ml/min. The concentration of methanol was 0% for 0-5 min and 0-5% linear gradient for 5-35 min.
The column temperature was maintained at 40 °C by a column oven, and
eluents were monitored at 280 nm.
Treatments with Repair Enzymes--
25F was 5'-end-labeled as
described for the primers (see below) and annealed to the complementary
strand (25COM). 25F/25COM (0.02 pmol) was incubated with AlkA (1 pmol)
followed by endonuclease (Endo) IV (0.03 pmol) in a reaction buffer (10 µl) at 37 °C. Alternatively, the substrate was incubated with Endo
III (0.3 pmol), formamidopyrimidine glycosylase (Fpg, 0.3 pmol), or
Endo IV (0.03 pmol) in a similar manner. The procedures of the
enzymatic treatments were essentially similar to those reported
previously for AlkA (28, 37), Endo III (38), Fpg, and Endo IV (39).
After incubation, products were analyzed by 16% denaturing PAGE.
Preparation of Template-Primer--
The primers were
5'-end-labeled using T4 polynucleotide kinase and
[ Analysis of Translesion DNA Synthesis--
The annealed
template-primer (0.5 pmol) containing T (24AT/P10 and 24CT/P10) or fU
(24AF/P10 and 24CF/P10) at the same position was incubated with Pol I
Kf (exo Analysis of Nucleotides Incorporated Opposite fU--
To
determine the nucleotide incorporated opposite fU, primer extension
reactions were performed in a manner essentially similar to that
described for translesion DNA synthesis except that the reaction
mixture contained a single dNTP (50 µM) and incubation time was 5 min. The template-primer used in the analysis was 24AF/13T, 24GF/13C, 24CF/13G, and 24TF/13A that contained fU at the same position
and different nearest base pairs next to fU (i.e. primer terminus base pairs). For control reactions, primer extension assays
were also performed using template-primers (24AT/13T, 24GT/13C, 24CT/13G, and 24TT/13A) that contained T in place of fU. By using these
template-primers containing fU or T, kinetic parameters of nucleotide
incorporation opposite fU and T were also determined. For dATP
incorporation, the dATP concentration was 0.05-1 µM, and
the amount of Pol I Kf (exo
The pH effect on the incorporation of dATP and dGTP opposite template
fU or T was determined by varying the pH (pH 7.2-8.6) of the
polymerase reaction buffer as described above. The template-primer (24AF/13T and 24AT/13T, 0.5 pmol) was incubated with Pol I Kf (exo Extension of Mismatched Primer Termini--
Template-primers
(0.5 pmol) containing a correctly paired (24AT/14TA and 24AF/14TA) or
mismatched (24AT/14TG and 24AF/14TG) primer terminus were incubated
with Pol I Kf (exo Electrophoresis--
DNA polymerase reactions were terminated by
adding loading buffer containing 0.1% xylene cyanol, 0.1% bromphenol
blue, 20 mM EDTA, and 95% formamide. The sample was boiled
and subjected to 16% denaturing polyacrylamide gel electrophoresis
(PAGE). Electrophoresis was performed at 1800 V, and gel was
autoradiographed at Nucleoside Composition of Oligonucleotides--
To ensure the
validity of phosphoramidite method used in the present preparation of
oligonucleotides containing fU, pilot oligonucleotides containing T
(25T) and fU (25F) in the same sequence were prepared. 25T and 25F were
digested by nuclease P1 and alkaline phosphatase, and the nucleoside
composition was analyzed by HPLC. Digestion of 25T resulted in the HPLC
peaks of dC, dG, dT, and dA with an expected molar ratio (8:6:4:7)
(data not shown). In the HPLC analysis of the digested 25F, two extra
peaks were observed in addition to the four normal nucleosides (Fig.
1A). The first peak eluted at
15.1 min was readily identified as 5-formyl-2'-deoxyuridine (fdU) by
comparison with the retention time of authentic fdU. The peak at 32.4 min (indicated by *) was an unknown product. When authentic fdU was
incubated under the same conditions as those used for oligonucleotide
digestion, fdU was partially converted to this product (Fig.
1B). The formyl group of fdU is fairly reactive and forms
adducts with nucleophiles (40). Accordingly, this product is most
likely an adduct between fdU and a nucleophilic molecule present in the
reaction buffer or enzyme preparations. The long HPLC retention time of
the product relative to fdU and retention of the UV absorption around
280 nm were also consistent with the adduct formation of the exocyclic
formyl group of fdU. The fU moiety of fdU is known to be degraded by
strong base and oxidizing reagents, giving rise to ring fragmentation
products (27, 41) and 5-carboxyuracil (42), respectively. If such products were formed during the preparation of 25F, they might be
present as contaminated lesions in 25F. However, the retention of the
UV absorption (around 280 nm) of the product was inconsistent with the
ring fragmentation products bearing no chromophores. That retention
time of the product (32.4 min) was much longer than fdU (15.1 min) in
the reversed phase HPLC column also contradicted the expected very
short retention time of 5-carboxy-2'-deoxyuridine bearing a negative
charge of a carboxylate ion. Thus, the product was not the ring
fragmentation products or 5-carboxy-2'-deoxyuridine. Moreover, when the
amount of authentic fdU converted to the putative adduct was taken into
account, the corrected molar ratio of nucleosides in 25F agreed with
the expected value (dC:dG:dT:dA:fdU = 8:6:3:7:1).
We also attempted to identify the structure of the unknown product by
mass spectrometric analysis after isolating the product by HPLC.
However, the attempt was unsuccessful because of the lack of the
apparent molecular ion (M+) in the spectrum. In an
alternative approach, 25F (as a duplex) was digested by several DNA
repair enzymes with different damage specificities. Consistent with the
previous reports (28, 37), the treatment with AlkA followed by Endo IV
resulted in incision of 25F at the fU site (data not shown). In
contrast, neither Endo III, Fpg, nor Endo IV incised
25F,2 supporting the absence
of base damage other than fU in 25F. On the basis of the results from
the composition analysis and the treatment with repair enzymes, we
concluded that fU was successfully incorporated into oligonucleotides
in the present procedure of synthesis.
Translesion DNA Synthesis at the fU Site--
To clarify whether
fU present in template DNA constitutes a replication block, 24AF and
24AT containing fU and T, respectively, at the same site (4 nucleotides
beyond the primer terminus) were primed by 32P-labeled P10,
and the templates were replicated by Pol I Kf (exo Nucleotides Incorporated Opposite fU--
Since efficient
translesion DNA synthesis occurred at the fU site, the nucleotide
incorporated opposite this lesion was analyzed by a primer extension
assay. In this assay, primers (13T, 13C, 13G, and 13A) that were 1 nucleotide shorter than the template fU site were annealed to
appropriate templates (24AF, 24GF, 24CF, and 24TF), and the primers
were extended by Pol I Kf (exo Parameters of dAMP and dGMP Incorporation--
For quantitative
analysis of the nucleotide incorporation efficiency and the sequence
context effect, kinetic parameters of dAMP and dGMP incorporation
opposite fU and T were determined by the gel fidelity assay under
standing start conditions (43). The experiments were performed as
described under "Experimental Procedures" using a set of
template-primer employed above (see under "Nucleotides Incorporated
Opposite fU"). Table II summarizes the
parameters (Vmax and Km) and
efficiencies
(fA = Vmax/Km)
of dAMP incorporation (average of two experiments). Although there were
variations depending on the nearest neighbor base pair, the
Vmax values for dAMP incorporation were
consistently higher for T than fU. Conversely, the
Km values for T were consistently lower than for fU.
Consequently, the incorporation efficiency of dAMP
(fA) opposite fU was reduced to 1/4 to
1/2 of that opposite T. The efficiency difference between T and fU was not large but significant, showing that conversion of T to fU in
template DNA slows down incorporation of the correct nucleotide dAMP.
Table III summarizes the parameters
(Vmax and Km) and
efficiencies (fG = Vmax/Km) of dGMP
misincorporation together with the replication error frequencies (fRE = fG/fA)
(average of two experiments). With the same nearest neighbor base pair,
the Vmax values for dGMP misincorporation were
consistently higher for fU than T. However, the Km
values for fU showed no systematic variations. Despite these
variations, misincorporation of dGMP was favored for fU over T by
1.1-5.6-fold as judged from relative fG. Granting
competitive incorporation of dAMP and dGMP at the same site (T or fU)
of DNA, the replication error frequency
(fRE = fG/fA)
was calculated for individual template-primers using the data in Tables
II (fA) and III (fG). The values
of fRE for fU were consistently in a
10 pH Effects of dAMP and dGMP Incorporation Opposite
fU--
We have previously reported pH-dependent
misincorporation of fdUTP opposite template G by DNA polymerase, and we
have pointed out the importance of an ionized (or enolate) form of fU
in fU:G mispair formation (27). To ask whether this mispairing scheme also held for fU in template DNA, the pH effect on the dGMP
misincorporation was analyzed. Template-primers containing fU
(24AF/13T) or T (24AT/13T) were incubated with Pol I Kf
(exo Extension of Matched and Mismatched Primer Termini--
The primer
extension assay described above revealed that fU in template DNA
directed incorporation not only of correct dAMP but also of incorrect
dGMP, although less efficiently, during DNA synthesis, thereby giving
rise to matched (fU:A) and mismatched (fU:G) primer termini. It is
known that mismatched primer termini are extended less efficiently than
matched termini by DNA polymerases and constitute a barrier for
erroneous replication of DNA. Thus, the extension of primer termini
containing fU:A and fU:G pairs was examined, and the results were
compared with those of T:A and T:G pairs. Fig.
7 shows gel data when matched (24AT/14TA
(T:A pair) and 24AF/14TA (fU:A pair)) and mismatched (24AT/14TG (T:G pair) and 24AF/14TG (fU:G pair)) primer termini were extended by Pol I
Kf (exo fU is one of the major oxidative thymine lesions found in DNA and
nucleoside that were exposed to ionizing radiation (44-46), Fenton-type reactions (46, 47), photosensitized reactions (48, 49), and
peroxy radicals (50). The yield of fU in Fenton-type reactions and
The mismatch extension frequency (fEX) in the
absence of proofreading depends explicitly on the binding constant of
DNA polymerase to matched versus mismatched template-primer DNA as well as on the concentrations of the template-primer DNA and
next correct dNTP (60). Accordingly, to evaluate the intrinsic mismatch
extension frequency (fEX(int)), possible
differential binding of DNA polymerase to matched and mismatched primer
termini needs to be taken into account under standing start conditions, i.e. differential binding to T:A versus T:G and
fU:A versus fU:G termini in this study. The relationship
between fEX and fEX(int) has been formulated in Equation 1 (60), where [Dr] and [Dw]
are the concentrations of template-primer DNA having correctly and
incorrectly paired primer termini (Dr and
Dw), respectively, and Kr and
Kw are the equilibrium constants for dissociation of
polymerase-Dr and polymerase-Dw
complexes, respectively.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C-6 saturation products such as thymine glycol
(5,6-dihydroxy-5,6-dihydrothymine) and 5,6-dihydro-5-hydroxythymine.
5,6-Dihydrothymine belongs to this group, although it is a reduction
product formed by ionizing radiation. The second group is ring
fragmentation products such as a urea residue and its analogues. The
response of DNA polymerases to the first and second groups has been
clarified fairly well by in vitro and in vivo
studies (13-15, 22, 23). The third group includes
5-hydroxy-5-methylhydantoin, a ring contraction product. The ability of
this lesion to block DNA replication has been demonstrated recently
(24) by in vitro DNA polymerase reactions using a defined
oligonucleotide template. The fourth group contains methyl oxidation
products such as 5-hydroxymethyluracil and 5-formyluracil (fU).1 Several lines of
evidence indicate that 5-hydroxymethyluracil is neither a replicative
block nor mutagenic (25, 26) and hence is an innocuous lesion.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (110 TBq/mmol) was
purchased from Amersham Pharmacia Biotech. Escherichia coli
DNA polymerase I Klenow fragment (Pol I Kf), Pol I Kf deficient in
3'-5'-exonuclease [Pol I Kf (exo
)], and T4
polynucleotide kinase were obtained from New England Biolabs, and
Penicillium citrium nuclease P1 and calf intestine alkaline
phosphatase were from Roche Molecular Biochemicals.
List of oligonucleotides used in this study
-32P]ATP. Appropriate template and primer (molar
ratio = 2:1) were annealed in 20 mM Tris-HCl (pH 7.5)
and 50 mM NaCl by heating the solution at 90 °C for 5 min and allowing to cool slowly to room temperature.
) (0.05 unit) and four dNTPs (50 µM
each) in a polymerase reaction buffer (15 µl) at 25 °C for 3-10
min. The polymerase reaction buffer consisted of 66 mM
Tris-HCl (pH 7.5), 1.5 mM 2-mercaptoethanol, and 6.6 mM MgCl2.
) was 0.002 unit, whereas for
dGTP incorporation, those were 10-80 µM and 0.03 unit.
The incubation time was 5 min for both dATP and dGTP incorporation
assays. Under these conditions, the extent of primer elongation was
essentially proportional to the reaction time and the unit of Pol I Kf
(exo
) used. The initial velocity of the reaction
(V) (average of two experiments) was calculated as the
percentage of the extended primer per min per 0.03 unit of Pol I Kf
(exo
). Km and
Vmax values were evaluated from a hyperbolic curve fitting program.
) (0.03 unit) and dATP or dGTP (20 µM)
at 25 °C for 5 min. For a wide pH range (pH 6.9-9.3), GTA buffer
(buffering capacity pH 3.5-10) was used in place of the Tris buffer
for the DNA polymerase reaction. The composition of GTA buffer was
3,3-dimethylglutaric acid, Tris, and 2-amino-2-methyl-1,3-propanediol
(17.3 mM each), and pH was adjusted by adding HCl or NaOH.
) (0.05 unit) and dCTP (10 µM) in the polymerase reaction buffer (15 µl) at
25 °C for 5 min. Alternatively, kinetic parameters of the reaction
(average of two experiments) were determined by varying the dCTP
concentration between 0.01 and 1 µM for the correctly paired primer terminus (24AT/14TA and 24AF/14TA) or 0.01-20
µM for the mispaired primer terminus (24AT/14TG and
24AF/14TG). The reaction was performed as described above except that
the amount of Pol I Kf (exo
) was 0.015 unit. The
parameters for mismatch extension with templates 24CT and 24CF were
also determined in the same manner.
80 °C overnight. The radioactivity of the
separated bands was quantified on a PhosphorImager Fuji Bas 2000.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HPLC analysis of nucleosides in 25F.
A, 25F containing fU was digested by nuclease P1 and
alkaline phosphatase as described under "Experimental Procedures."
An aliquot of the reaction mixture was analyzed by reversed phase HPLC
equipped with a C18 WS-DNA column (4.6 × 150 mm). The sample was
eluted by a gradient of methanol (0% for 0-5 min and 0-5% linear
gradient for 5-35 min) in 10 mM sodium phosphate buffer
(pH 7.4). The flow rate was 0.8 ml/min, and the monitoring wavelength
was 280 nm. The attribution of the elution peak is indicated
above the peak. The elution peak with an asterisk
was a putative fdU adduct formed by the enzymatic treatment.
B, authentic fdU was treated by nuclease P1 and alkaline
phosphatase and subjected to HPLC analysis as described above. Note
that fdU before incubation was eluted as a single peak (not shown) and
no adduct (*) was observed.
) for
up to 10 min. The resulting products were analyzed by denaturing PAGE
(Fig. 2). After 3 min of incubation, the
primer annealed to the undamaged template 24AT was almost completely
extended to a fully replicated product (lane 2). Similarly,
the primer annealed to 24AF containing fU was mostly extended to fully
replicated and 1 nucleotide shorter products after 3 min of incubation
(lane 6). In addition, very weak bands also appeared at and
1 nucleotide before the fU site, indicating a pause of DNA synthesis at
these sites. Quantification of the arrested and bypassed products
showed that 91% of the original primer was extended beyond the fU site at 3 min. This result indicates that fU in template DNA allows efficient translesion DNA synthesis. Similar results were obtained with
templates 24CF and 24CT (data not shown).
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Fig. 2.
Translesion DNA synthesis at the fU
site. DNA templates containing T (24AT) and fU (24AF) at the same
site were primed with 32P-labeled P10. 24AT/P10 and
24AF/P10 (0.5 pmol) was incubated with Pol I Kf (exo )
(0.05 unit) and four dNTPs (50 µM each) at 25 °C, and
products were analyzed by 16% denaturing PAGE. Templates (24AT and
24AF) and incubation times (3, 5, and 10 min) are indicated on the
top. Lanes 1 and 5, template-primer
without the polymerase reaction; lane 9, 14-mer marker
(32P-labeled 14TA in Table I) showing the position of
fU.
) in the presence of a
single dNTP (50 µM) at 25 °C for 5 min. These
template-primers (24AF/13T, 24GF/13C, 24CF/13G, and 24TF/13A) contained
four different nearest neighbor base pairs in the primer terminus.
Control experiments were also performed under the same conditions using
template-primers (24AT/13T, 24GT/13C, 24CT/13G, and 24TT/13A) that
contained T instead of fU. The reaction products were analyzed by
denaturing PAGE. Fig. 3A shows
PAGE data obtained for 24AF/13T and 24AT/13T containing an A
(template):T (primer) pair at the primer terminus. According to the
band intensity of the extended product (14-mer), dAMP was most
efficiently incorporated opposite fU (lane 6) as well as T
(lane 1), with a preference for T. The bands indicative of
misincorporation of dGMP opposite fU (lane 7) and T
(lane 2) were also observed, but the incorporation was more
efficient for fU than T. In the presence of dCTP and dTTP, extended
products were not observed over the background for both templates
containing fU (lanes 8 and 9) and T (lanes 3 and 4), showing that the misincorporation frequency
of dCMP and dTMP was below the detection limit under these conditions. Essentially similar results were obtained with other template-primers containing G:C (Fig. 3B), C:G (Fig. 3C), and T:A
(Fig. 3D) as the nearest neighbor base pairs. Accordingly,
fU directed incorporation of correct dAMP and to a less extent
incorrect dGMP but not pyrimidine nucleotides (dCMP and dTMP).
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Fig. 3.
PAGE analysis of the nucleotide incorporated
opposite fU. Template-primers (0.5 pmol) containing four different
primer terminus base pairs were incubated with the indicated dNTP (50 µM) and Pol I Kf (exo ) (0.05 unit) at
25 °C for 5 min, and the incorporated nucleotide opposite
X (= T or fU) was analyzed by 16% denaturing PAGE.
Template-primer used was 24AT/13T (lanes 1-4) and 24AF/13T
(lanes 6-9) (A), 24GT/13C (lanes
1-4) and 24GF/13C (lanes 6-9) (B),
24CT/13G (lanes 1-4) and 24CF/13G (lanes 6-9)
(C), 24TT/13A (lanes 1-4) and 24TF/13A
(lanes 6-9) (D). A-D, lane
5 shows template-primer without the polymerase reaction. The
arrow indicates the extended primer. The sequence
surrounding T or fU (indicated by X) is shown
above the gel.
4 range, whereas those for T were in a
10
5 range. Thus, the increase in
fRE due to the substitution of fU for T was
3.1-14.3-fold (Fig. 4A).
These increases arose from reduced fA (Table II) and
increased fG (Table III). Comparison of the
parameters (Vmax and Km) in
Tables II and III also indicated that discrimination of the nucleotide
at the fU site originated from Vmax and
Km. The averaged discrimination factors for
Vmax and Km were 16 and 360, respectively, which were calculated from the ratio of the parameters
for dAMP versus dGMP incorporation. Therefore, the
contribution of Km was much greater than that of Vmax.
Parameters (Vmax and Km) and efficiencies (fA)
of dAMP incorporation opposite template T and fU
Parameters (Vmax and Km) and efficiencies (fG)
of dGMP misincorporation opposite template T and fU, and replication
error frequencies (fRE)
View larger version (24K):
[in a new window]
Fig. 4.
Sequence context effects on
fA, fG,
fRE, and increases in
fRE and fEX
associated with conversion of T to fU. A, increases in the
replication error frequency (fRE) associated with
conversion of T to fU. The ratio of the replication error frequencies
(fRE (X = fU)/fRE
(X = T)) was calculated for the same 3'-nearest
neighbor base using the data in Table III. The ratio was plotted
against the template sequence (3'-NX-5', N = A, G, C, and T,
X = T and fU). B, increases in the mismatch
extension frequency (fEX) associated with conversion
of T to fU. The ratio of the misextension frequencies
(fEX (X = fU)/fEX
(X = T)) was calculated for the corresponding templates
(i.e. 24AF versus 24AT and 24CF versus
24CT) using the data in Table IV. The ratio was plotted against the
template sequence (3'-NXG-5', N = A and C,
X = T and fU). C, sequence context effects
on the incorporation efficiencies of dAMP (fA).
D, sequence context effects on the misincorporation
efficiencies of dGMP (fG). E, sequence
context effects on the replication error frequency
(fRE). C-E, the values of
fA, fG, and
fRE were taken from Tables II and III and plotted
against the template sequence (3'-NT-5' and 3'-NF-5', N = A, G, C,
and T, F = fU).
) and a single dNTP (dGTP or dATP, 20 µM) at pH 7.2-8.6. The percentage of the extended primer
resulting from incorporation of dAMP or dGMP was determined by PAGE
analysis (Fig. 5). Incorporation of dAMP
was virtually unaffected by the pH change and was less efficient for fU
than T (Fig. 5, A and C). In contrast,
incorporation of dGMP opposite fU showed a clear pH dependence and the
amount of dGMP increased with increasing pH (Fig. 5, B and
D). Although dGMP incorporation opposite T was also
pH-dependent, the increase with pH was extremely small
(Fig. 5, B and D). The pH effect on dGMP
misincorporation opposite fU was further analyzed in a wider pH range
(pH 6.9-9.3) using GTA buffer in place of Tris buffer (Fig. 5D,
inset). The plot of the efficiency of dGMP misincorporation against pH showed a sigmoidal curve reminiscent of a pH titration, although the efficiency was somewhat different between the GTA and Tris
buffer systems. Since the pKa values of fU and T
were 8.6 and 10.0, respectively, these results strongly suggest that an
acid-base equilibrium of fU (Fig.
6A) is involved in the misincorporation of dGMP, and the ionized (or enolate) form of fU forms
a mispair with incoming dGTP during DNA synthesis (Fig. 6B,
left).
View larger version (38K):
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Fig. 5.
pH effects on the incorporation of dAMP and
dGMP opposite fU. Template-primers (24AF/13T and 24AT/13T, 0.5 pmol) were incubated with Pol I Kf (exo ) (0.03 unit) in
the presence of dATP or dGTP (20 µM) at pH 7.2-8.6 (Tris
buffer) or 6.9-9.3 (GTA buffer). Incubation was performed at 25 °C
for 5 min. Incorporation of dAMP (A) and dGMP (B)
opposite template fU and T (indicated above the gel) was
analyzed by 16% denaturing PAGE. A and B, pH of
the reaction mixture was 7.2 (lanes 1 and 6), 7.7 (lanes 2 and 7), 8.0 (lanes 3 and
8), 8.3 (lanes 4 and 9), and 8.6 (lanes 5 and 10). The extended primer is
indicated by the arrow. The percentage of the extended
primer resulting from incorporation of dAMP (C) and dGMP
(D) was plotted against pH based on the product analysis in
A and B. dGMP misincorporation opposite fU was
also measured in a wide pH range (pH 6.9-9.3) using GTA buffer, and
the result is shown in the inset of D. C and D, incorporation opposite template T and fU
is represented by open and closed symbols,
respectively.
View larger version (18K):
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Fig. 6.
A proposed mechanism for mispair formation
between fU and G. A, an acid-base equilibrium of fU
(right) involving keto and ionized (enolate) forms and a
tautomeric equilibrium of fU (left) involving keto and enol
forms. B, base pairing schemes for the fU:G mispair
involving ionized (or enolate) and keto forms of fU.
) in the presence of dCTP, a nucleotide to be
incorporated following the primer termini. The matched primer termini
containing T:A and fU:A pairs were elongated with comparable
efficiencies (lanes 2 and 4). Although extension
of the mismatched primer termini containing T:G (lane 6) and
fU:G (lane 8) pairs were less efficient than matched ones,
the extension of the fU:G terminus was clearly preferred over T:G. For
quantitative comparison of the extension efficiencies, kinetic
parameters (Vmax and Km) for
dCMP incorporation were determined by the gel fidelity assay under standing start conditions (43) using 24AT, 24AF, 24CT, and 24CF (average of two experiments) (Table IV).
Irrespective of the undamaged (24AT and 24CT) or damaged (24AF and
24CF) templates, the matched primer termini containing fU:A or T:A
pairs were efficiently extended, with a slight preference of T:A
(~1.2-fold as the extension efficiency (fC)). With
the matched termini, Km values of extension
(i.e. dCMP incorporation) were comparable to those of dAMP
at the previous site (Table II), whereas the corresponding Vmax values were severalfold lower, presumably
due to the difference in the incorporated nucleotide (C or A) or the
sequence context. The extensions of the mismatched primer termini
containing fU:G and T:G were inefficient, and the extension efficiency
(fC) was 2 or 3 orders of magnitude lower than that
of the corresponding matched termini (Table IV). For both T and fU,
discrimination of the matched and mismatched termini exclusively
originated from Km. Interestingly, the mismatched
primer termini containing the fU:G pair was extended with significantly
higher efficiencies than those containing the T:G pair. The mismatch
extension frequency (fEX = fC(mismatched
terminus)/fC(matched terminus) for the same
template) was in a 10
2 range for fU, whereas
that for T was in a 10
3 range. The increase
in fEX associated with the substitution of fU for T
was 8.2- (24AF versus 24AT) and 11.3-fold (24CF
versus 24CT) (Fig. 4B). Accordingly, conversion
of T to fU in template DNA promotes not only misincorporation of dGMP
(Fig. 4A) but also elongation of the resulting mismatched
primer termini (Fig. 4B).
View larger version (26K):
[in a new window]
Fig. 7.
PAGE analysis of the extension of mismatched
primer termini containing T:G and fU:G pairs. Template-primers
(0.5 pmol) containing correctly paired (24AT/14TA (T:A pair) and
24AF/14TA (fU:A pair)) and mismatched (24AT/14TG (T:G pair) and
24AF/14TG (fU:G pair)) primer termini were incubated with Pol I Kf
(exo ) (0.05 unit) in the presence of dCTP (10 µM) at 25 °C for 5 min. Products were analyzed by 16%
denaturing PAGE. Lanes 1, 3, 5, and 7 show
template-primers without the polymerase reaction, and lanes 2, 4, 6, and 8 show the extended products formed by the
polymerase reaction. The base pair in the primer terminus is indicated
above the gel.
Parameters (Vmax and Km) and efficiencies
(fC) of extension of matched and mismatched primer
termini, and mismatch extension frequencies (fEX)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiation is comparable to those of 8-oxoG (47) and
5-hydroxypyrimidines (45) that are known as major mutagenic oxidative
base lesions (51-55). Bacterial (28, 37, 56) and mammalian (57, 58)
cells contain repair enzyme or activity that excises fU from damaged
DNA, implying potential genotoxic influences of this lesion in
vivo. Direct incorporation of fdUTP into permeated E. coli cells resulted in a small but significant increase in
chromosomal lacI mutation with G:C
A:T transitions being
most preferred (59). fdU was also mutagenic to Salmonella typhimurium when added to the culture medium (44). In the present study, we have assessed the genotoxic potential of fU in template DNA
by utilizing in vitro DNA replication reactions. The product analysis of translesion synthesis revealed that fU constituted very
weak blocks to DNA synthesis (Fig. 2). Thus, unlike other thymine
lesions such as thymine glycol, urea residues, and
5-hydroxy-5-methylhydantoin (13-15, 24), fU will exert a weak to
negligible cytotoxic effect due to inhibition of DNA replication
in vivo. Conversely, fU was shown to be a potentially
mutagenic lesion based on the following results. First, the
substitution of T by fU promoted misincorporation of incorrect dGMP
(1.1-5.6 times as fG) and at the same time retarded
incorporation of correct dAMP (1/4 to 1/2 as
fA), hence leading to 3.3-14.3-fold increases in
the replication error frequency (fRE) relative to T
(Fig. 4A). Second, the resulting mismatched primer terminus
containing an fU:G pair was more readily extended (8.2-11.3 times as
fEX) than that containing a T:G pair (Fig.
4B) (see also the discussion below on
fEX). This step will affect the probability that a genome DNA molecule is replicated to completion, and thereby
scored as mutation. According to the present data, fU is moderately
mutagenic, but for more quantitative estimation of the mutation
frequency of fU, it is necessary to consider the influence of repair
and the property of replicative DNA polymerases.
In Equation 1, it is generally assumed that the affinity of DNA
polymerase for a correctly paired terminus is similar to or higher than
that for an incorrectly paired terminus (Kw
(Eq. 1)
Kr). The values of
[Dr] and [Dw] are both 33 nM in this study. When Pol I (exo
) has
comparable affinities (Kr
Kw)
for the matched and mismatched termini (i.e. T:A
and T:G, and fU:A and fU:G), fEX in Table
IV approximately represents the intrinsic value. According to
Equation 1, the largest discrepancy between fEX
and fEX(int) occurs when
Kw is much higher than [Dw]
(Kw
33 nM). In this case, Equation 1
can be transformed into Equation 2 by approximation.
Although the Kr values of Pol I
(exo
(Eq. 2)
) are not known, those for Avian
myeloblastosis reverse transcriptase and Drosophila melanogaster DNA polymerase
have been estimated as 5 and
20-50 nM, respectively (60). Granted that the
Kr value of Pol I (exo
) is in a
similar range (5-50 nM), the fEX values
in Table IV are subjected to a 7.6-fold reduction. However, the
correction factor given by Equation 2 is presumably similar for the T
and fU templates since the affinities (Kr) of Pol I
(exo
) for the structurally resembling T:A and fU:A
termini are likely comparable. These considerations suggest that the
relative difference in fEX(int) for the
T versus fU templates remains similar to that shown in Fig.
4B, although the absolute value of
fEX(int) may be lower than that obtained
experimentally in this study (fEX).
Concerning the mispairing mechanism of fU, we have previously suggested
participation of the ionized (or enolate) form of fU based on the
pH-dependent misincorporation of fdUTP opposite template G
(27). Privat and Sowers (61) also proposed a similar mispairing scheme
on the basis of pKa measurement of fdU and related
nucleosides. Consistent with this mechanism, the efficiency of dGMP
misincorporation opposite template fU increased around the
pKa value of fU (pKa = 8.6),
whereas the corresponding increase for T (pKa = 10.0) was much smaller than that for fU (Fig. 5, B and
D). The result obtained for T also agrees with a small
increase in the base substitution frequency of Pol I
(exo) in this pH range (62). Unlike thymine bearing an
electron donating methyl group, fU has an electron withdrawing formyl
group that promotes ionization of fU in an acid-base equilibrium (Fig. 6A). According to the pKa values, the
fraction of the ionized form of fU increases from 4 to 50% in
the pH range of 7.2-8.6 but that of thymine is virtually negligible
(0.2-4%). Ionized fU can form a base pair with incoming dGTP through
two hydrogen bonds (Fig. 6B, left). The base pair formed
between ionized fU and dGTP essentially assume Watson-Crick geometry
(or B form geometry) and can fit into the active site of DNA
polymerase. Since the geometric recognition is key to discrimination of
correct versus incorrect nucleotides by DNA polymerases (1),
this geometry probably promoted misincorporation of dGMP opposite fU.
Participation of base ionization promoted by electron withdrawing
substituents has been demonstrated in the mispairing of 5-halogenated
uracils (BrU and 5-fluorouracil) with G (32). Thus, fU and
5-halogenated uracils share a common mutation mechanism. Although
participation of a rare enol tautomer of fU (Fig. 6A) in the
mispairing with G cannot be fully ruled out, recent NMR studies show
that the tautomeric equilibrium between keto and enol forms of fU is
not significantly affected by oxidation of the methyl group of T to the
formyl group (63, 64). Therefore, involvement of the enol form of fU is
unlikely in the mispairing with G. It is assumed that after dGMP
incorporation, the resulting fU(ionized):G pair in Watson-Crick
geometry shifts to wobble geometry (fU(keto):G) due to the acid-base
equilibrium (Fig. 6B, right). However, a certain fraction of
the base pair will still exist as an fU(ionized):G pair whose geometry
can again promote incorporation of the next nucleotide. Probably this
is the reason why the mismatched primer terminus containing an fU:G
pair was more efficiently extended than that containing a T:G pair.
Although there is no experimental evidence that directly shows the
equilibrium between fU(keto):G and fU(ionized):G base pairs in duplex
DNA, the presence of such a equilibrium has been demonstrated for
BrU(keto):G and BrU (ionized):G pairs in a duplex oligonucleotide by
the NMR study (65). According to the proposed mutation mechanism for
fU, it is reasonable that 5-hydroxymethyluracil, another methyl
oxidation product of T, does not direct misincorporation (25, 26) since
the hydroxymethyl group has electron donating nature and cannot promote
ionization of the base. Although a mutation mechanism involving an
altered acid-base equilibrium has been previously demonstrated for
5-halogenated uracils (32), to our knowledge, fU is the first example
adapting to this mechanism among oxidative DNA base lesions.
To assess the sequence context effect on the base pairing property of
fU, the 3'-nearest neighbor base of template fU and the paired base
(i.e. the primer terminus base pair) was systematically changed, and the nucleotide incorporated opposite fU was analyzed. fU
with the four possible nearest neighbor base pairs directed incorporation of dAMP and to a less extent dGMP but not dCMP and dTMP
(Fig. 3). Thus, 3'-nearest neighbor base exhibited no influence concerning the type of base pairs formed from template fU. Combining the result with fdUTP (27, 28) and template fU (this study), it follows
that fundamental base pairing symmetry is retained whether the fU base
pairs are formed from incoming fdUTP or template fU during DNA
polymerase reactions. Although fundamental base pairing symmetry held
with respect to the formation of the fU base pairs, the nearest
neighbor base pair showed quantitative effects on the incorporation
efficiency of dAMP and dGMP. Regardless of the correct or incorrect
incorporation (Fig. 4, C and D), the sequence
context effect was less pronounced for fU than T. For example, the
difference in fG for T was 9.4-fold for the
highest (TT) and lowest (GT) sequences, whereas the corresponding value
(TF versus AF, F = fU) was only 2.2-fold for fU (Fig.
4D). Another notable sequence context effect was a tendency
of preferred incorporation of dAMP and dGMP for the sequences
containing 3'-pyrimidines over 3'-purines, although the difference was
not so large. In other words, incorporation of the nucleotides was
favored for the primer terminus containing purines. This was common to
T and fU. Presumably, stabilization of the incoming purine nucleotides through a favored purine-purine stacking interaction
(purine-purine > pyrimidine-purine > pyrimidine-pyrimidine) (66) promoted their incorporation over
pyrimidine nucleotides. Despite the sequence context dependent
variations of fA and fG, the fRE (=fG/fA)
values of fU were consistently higher than those of T and were
virtually independent of the nearest neighbor base pair (Fig.
4E). This result suggests that the distribution of T C
transitions induced by fU will be relatively uniform with respect to
the variation of the 3'-nearest neighbor base unless heterogeneous
formation or repair of fU occurs in cells. Finally, the relative
increase in fRE associated with the conversion of T
to fU was calculated. The order of the increase was A > G > C > T with respect to the 3'-nearest neighbor base (Fig.
4A), showing an inverse correlation with that (T > C > G > A) ranged by fRE of T (Fig.
4E). Thus, the sequence with a low replication error
frequency before conversion to fU gave rise to a relatively large
increase in the replication error frequency after conversion to fU.
Previously, Zhang et al. (29, 30) assessed the mutagenic
potential of fU using an in vitro system similar to that
used in this study. According to their experiments with Pol I Kf, Pol I
Kf (exo), and thermophilic DNA polymerases, fU directed
incorporation of dCMP as well as dAMP in all cases but not dGMP and
dTMP at all. The incorporation efficiency of dCMP relative to dAMP was 0.09-0.15 for Pol I Kf, 0.06 for Pol I Kf (exo
), and
0.23-0.27 for Tth DNA polymerase. These values are unusually high as a
frequency of pyrimidine:pyrimidine mispair formation by DNA
polymerases. Generally, pyrimidine:pyrimidine pairs are too small to
fit into the B form helix. For this reason, formation of these mispairs
is not favored by prokaryotic and eukaryotic DNA polymerases. The most
common mispairs are G:T mispairs with observed frequencies between
10
2 and 10
4, and
the least common ones are pyrimidine:pyrimidine mispairs with
frequencies between 10
4 and
10
5 (1). The rate of misincorporation of dNMP
opposite T by Pol I Kf and Pol I Kf (exo
) also follows
this rule (dGMP
dCMP
dTMP) (62, 67). The measurement of the
melting temperature (Tm) of duplex oligonucleotides
containing an fU:N pair (N = A, G, C, T) revealed that
Tm decreased in the following order: fU:A > fU:G > fU:T
fU:C.3
This order indicates that the fU:C pair exerts the largest
destabilization effect on DNA among the four possible base pairs and
further suggests the least favored formation of this pair during DNA
replication. In addition, Zhang et al. (29) found
significant incorporation of dCMP but not dGMP opposite normal T (Table
V), suggesting a fundamental discrepancy
in the experimental set up used in the present and their studies. We
repeated this experiment using the same template-primer
(i.e. template 1 and primer 3 shown in Table V). However,
the reported result was not reproduced, and misincorporation of only
dGMP was detected (Table V). Thus, T in this sequence context was not
particularly prone to incorporate dCMP. We also repeated another
control experiment with Tth DNA polymerase under the reported
conditions (29, 30). Template 1 (see Table V for the sequence) was
annealed to a 9-mer primer (5'-TGCAGGTCG) and primer extension assays
were performed in the presence of a single dNTP at 74 °C for 5 or 10 min. Although they observed incorporation of dAMP opposite T under
these conditions, we did not see incorporation of any nucleotides. We
believe the present result is reasonable in light of the expected low
melting temperature of the 9-mer primer (Tm = 30 °C). It is very likely that the template-primer dissociated at
74 °C and was unable to serve as a substrate for Tth DNA polymerase
in the present experiment. For the same reason, the reported
incorporation of dCMP with the 9-mer primer by Tth DNA polymerase (29,
30) is very unlikely to occur at such a high temperature. In view of
the inconsistencies such as the contradiction against the general
preference of mispair formation by DNA polymerases and the lack of
reproducibility of the certain experimental results, we believe fU
directs misincorporation of dGMP but not dCMP. Therefore, whether fU is
in incoming dNTP or a template, base pairing symmetry in the nucleotide
selection by DNA polymerase and the mutagenesis mechanism involving
ionized fU hold, although the incorporation efficiency of dAMP and dGMP varies depending on the nearest neighbor base pair.
|
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ACKNOWLEDGEMENTS |
---|
We thank Akira Matsuda and Naoko Karino (Hokkaido University) for communicating the Tm data for the duplexes containing fU.
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Note Added in Proof |
---|
Recently, Miyabe et al. (69) have reported that the fU site-specifically incorporated into plasmid vectors induces mutations but does not direct misincorporation of dCMP when the plasmids are replicated in Escherichia coli.
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FOOTNOTES |
---|
* This work was supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan (to H. I.) and by JSPS Research Fellowships for Young Scientists (to A. M.).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.
To whom correspondence should be addressed. Tel./Fax:
81-824-24-7457; E-mail: ideh@hiroshima-u.ac.jp.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M008598200
2 Recently, the activity of Endo III and Fpg for fU was reported by Zhang et al. (68). Our study on the kinetic parameter revealed that their activity for fU was 2-3 orders of magnitude lower than their intrinsic substrates (thymine glycol for Endo III and 7,8-dihydro-8-oxoguanine for Fpg). Thus, the activity of the two enzymes for fU was negligible under standard assay conditions, and extremely large excesses of enzymes were required to detect the activity for fU (A. Masaoka, H. Terato, Y. Ohyama, and H. Ide, manuscript in preparation).
3 The UV melting curves (plots of A260 against temperature) were measured using the duplexes (total strand concentration 3 µM) of 25F and its complementary strand containing A, G, C, or T as the base opposite fU in 10 mM NaCl and 10 mM sodium cacodylate (pH 7.0). The Tm value was evaluated from the inflection point of the melting curve (A. Masaoka, H., Terato, Y. Ohyama, N. Karino, A. Matsuda, and H. Ide, manuscript in preparation).
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ABBREVIATIONS |
---|
The abbreviations used are:
fU, 5-formyluracil;
fdUTP, 5-formyl-2'-deoxyuridine 5'-triphosphate;
fdU, 5-formyl-2'-deoxyuridine;
BrU, 5- bromouracil;
Pol I Kf, Escherichia coli DNA polymerase I Klenow fragment;
Pol I Kf (exo), Pol I Kf deficient in 3'-5'-exonuclease;
PAGE, polyacrylamide gel electrophoresis;
Endo, endonuclease;
Fpg, formamidopyrimidine glycosylase;
HPLC, high-performance liquid
chromatography.
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REFERENCES |
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