(Received for publication, February 8, 1995; and in revised form, May 19, 1995)
From the
We found that hydroxylation occurs at the C-2 position of
adenine by oxygen radical treatment (Fe-EDTA) of dA,
dATP, and single- and double-stranded DNA. This oxidatively damaged
base, 2-hydroxyadenine, was produced 3-6-fold and 40-fold less
than 8-hydroxyguanine when monomers and polynucleotides, respectively,
were treated. To determine whether the damaged nucleotide,
2-hydroxydeoxyadenosine triphosphate (2-OH-dATP), is incorporated into
a growing DNA, and to reveal the kinds of nucleotides opposite which
2-OH-dATP is incorporated, calf thymus DNA polymerase
and the
Klenow fragment of Escherichia coli DNA polymerase I were used
in in vitro DNA synthesis in the presence of 2-OH-dATP. DNA
polymerase
incorporated the nucleotide opposite T and C in the
DNA template. On the other hand, in an experiment using the Klenow
fragment, incorporation of 2-OH-dATP was observed only opposite T.
Steady-state kinetic studies indicated that incorporation of 2-OH-dATP
by DNA polymerase
opposite T was favored over that opposite C by
a factor of only 4.5. These results indicate that 2-OH-dATP, an
oxidatively damaged nucleotide, is a substrate for DNA polymerases and
is incorporated incorrectly by the replicative DNA polymerase.
Oxygen radicals produced in cells damage DNA and nucleotides,
and these processes are probably involved in mutagenesis and
carcinogenesis. One of the oxidative DNA damage is 8-hydroxyguanine
(8-OH-Gua). ()8-OH-Gua is produced by treatment of DNA with
reagents that produce oxygen radicals(1) , and it induces
mainly G to T
mutations(2, 3, 4, 5, 6, 7) .
The mutT mutant of Escherichia coli, in which
A
T to C
G transversions are induced with high
frequency(8) , lacks the activity that hydrolyzes
8-OH-dGTP(9) . Together with the fact that DNA polymerases
incorporate 8-OH-dGTP as a substrate(7, 9) , it
appears that 8-OH-Gua is generated by incorporation of the damaged
nucleotide as well as by direct modification of G in DNA(1) .
A question that has arisen is whether other modified nucleotides
that are produced by oxygen radicals are incorporated by DNA
polymerases, and whether the incorporated nucleotides induce mutations.
We report here that when dA, dATP, and DNA were treated with oxygen
radicals (Fe-EDTA)(10) , hydroxylation at the
C-2 position of adenine occurred to form 2-OH-Ade (2-hydroxyadenine,
1,2-dihydro-2-oxoadenine, or isoguanine) and that the hydroxylation in
dATP and dA occurred more frequently than that in DNA. Although Switzer et al.(11, 12) observed that the Klenow
fragment of E. coli DNA polymerase I (KF) and other DNA
polymerases incorporate 2-OH-dATP (``d-iso-GTP'' in their
reports) opposite T in a template DNA, they studied 2-OH-Ade as a
component of additional base pairs in nucleic acids, and not as
oxidative damage. They did not investigate the possibility of the
incorporation opposite A and G (C was used as a control). Moreover,
they did not use mammalian enzymes, which are important for studies of
carcinogenesis. We describe the results of in vitro DNA
synthesis reactions with mammalian DNA polymerase
(pol
) and
KF in the presence of 2-OH-dATP.
Reactions catalyzed by KF were carried out in a buffer solution
containing oligonucleotide templates annealed with primers (0.05
µM), 50 mM Tris-HCl (pH 8.0), 8 mM MgCl, 5 mM 2-mercaptoethanol, various amounts
of dNTP(s), and enzyme in a total volume of 10 µl at 20 °C.
Experiments with pol
were conducted at 25 °C in a 10-µl
reaction mixture containing oligonucleotide templates annealed with
primers (0.05 µM), 20 mM Tris-HCl (pH 8.0), 5
mM MgCl
, 3.3 mM 2-mercaptoethanol, bovine
serum albumin (0.2 mg/ml), and various amounts of dNTP(s) and enzyme.
Reactions were stopped by the addition of termination solution (95%
formamide, 0.1% bromphenol blue, and 0.1% xylene cyanol). Samples were
heated at 95 °C for 3 min and were then applied to a 7 M
urea, 20% polyacrylamide gel. Autoradiograms were obtained with a Fujix
BAS 2000 bioimage analyzer.
Figure 1:
Separation of 2-OH-dATP by
reverse-phase HPLC. Fe-EDTA treatment and separation
by HPLC were done as described under ``Experimental
Procedures.'' The material eluted at 5.4 min was identified as
2-OH-dATP by UV spectra (inset).
The yields of 2-OH-dA and 2-OH-dATP were 800/10 dA and
650/10
dA, respectively, and were 3.6- and 5.7-fold less
than those of 8-OH-dG and 8-OH-dGTP (Table 1). On the other hand,
the formation of 2-OH-Ade was less efficient, about 40-fold less than
that of 8-OH-Gua, when double- and single-stranded DNA were treated
(9.7 and 17.7/10
dA, Table 1). Therefore, 2-OH-Ade
appears to be produced more efficiently in monomers than in
polynucleotides.
Figure 2:
Incorporation of 2-OH-dATP by Klenow
fragment. A, template 1 was annealed with P-labeled primer 1. The template-primer complex (0.05
µM) was treated with KF (0.1 units) in the presence of 50
µM of each of the dNTP(s) in a total volume of 10 µl
under the conditions described under ``Experimental
Procedures.'' The reaction mixtures were incubated at 20 °C
for 60 min and were processed as described under ``Experimental
Procedures.'' Lane1, untreated primer 1; lane2, in the presence of dATP; lane3, in the presence of dATP and dGTP; lane4, in the presence of dATP, dGTP, and dCTP; lane5, in the presence of dATP, dGTP, dCTP, and dTTP; lane6, in the presence of 2-OH-dATP; lane7, in the presence of 2-OH-dATP and dGTP; lane8, in the presence of 2-OH-dATP, dGTP, and dCTP; lane9, in the presence of 2-OH-dATP, dGTP, dCTP, and dTTP. B, the template-primer complex (0.05 µM) was
treated with KF (0.1 units) in the presence of 50 µM of
each of the three dNTPs, with or without 50 µM 2-OH-dATP,
in a total volume of 10 µl under the conditions described under
``Experimental Procedures.'' The reaction mixtures were
incubated at 20 °C for 30 min and were processed as described under
``Experimental Procedures.'' Lanes1 and 10, untreated primer 1; lane2, in the
presence of dGTP, dCTP, and dTTP; lane3, in the
presence of 2-OH-dATP, dGTP, dCTP, and dTTP; lane4,
in the presence of dATP, dCTP, and dTTP; lane5, in
the presence of 2-OH-dATP, dATP, dCTP, and dTTP; lane6, in the presence of dATP, dGTP, and dTTP; lane7, in the presence of 2-OH-dATP, dATP, dGTP, and dTTP; lane8, in the presence of dATP, dGTP, and dCTP; lane9, in the presence of 2-OH-dATP, dATP, dGTP, and
dCTP.
Figure 3:
Incorporation of 2-OH-dATP by DNA
polymerase . A, template 1 was annealed with
P-labeled primer 1. The template-primer complex (0.05
µM) was treated with pol
(1.4 units) in the presence
of 50 µM of each of the dNTP(s) in a total volume of 10
µl, under the conditions described under ``Experimental
Procedures.'' The reaction mixtures were incubated at 25 °C
for 120 min and were processed as described under ``Experimental
Procedures.'' Lane1, untreated primer 1; lane2, in the presence of 2-OH-dATP; lane3, in the presence of 2-OH-dATP and dGTP; lane4, in the presence of 2-OH-dATP, dGTP, and dCTP; lane5, in the presence of 2-OH-dATP, dGTP, dCTP, and dTTP. B, reaction conditions were the same as described above,
except that template 2 was used instead of template 1. Lane1, untreated primer 1; lane2, in the
presence of 2-OH-dATP; lane3, in the presence of
2-OH-dATP and dCTP; lane4, in the presence of
2-OH-dATP, dCTP, and dATP; lane5, in the presence of
2-OH-dATP, dCTP, dATP, and dTTP.
We recently found that DNA
polymerases (DNA polymerases and
and KF) misincorporated
dAMP opposite 2-OH-Ade in a template DNA(18) . However, we did
not detect the incorporation of 2-OH-dATP opposite A in template DNAs,
as shown in Fig. 2and Fig. 3. Since the nucleotide
sequence used in the previous study (5`-dGCTA*ATATTCCGTCAT-3`, where A*
represents 2-OH-Ade) was different from those used in this study, we
tested whether 2-OH-dATP was inserted opposite A in a template with the
same flanking sequences as used in the previous study (Table 2,
template 3). However, pol
did not insert 2-OH-dATP opposite A,
whereas the enzyme incorporated the nucleotide opposite T and C (data
not shown). The modified nucleotide was incorporated opposite T and C,
but not opposite A, even in the sequence where dAMP is inserted
opposite 2-OH-Ade.
In this study we found that 2-OH-dATP is efficient produced
from dATP by treatment with oxygen radicals and that it is incorporated
into DNA incorrectly. It is interesting to compare the efficiency of
incorporation of 2-OH-dATP into DNA with that of 8-OH-dGTP. The
relative V/K
values of 8-OH-dGTP opposite C and A are 4.4
10
and 7.2
10
,
respectively, with exonuclease-free KF(19) . The obtained
relative V
/K
values (the F
) of 2-OH-dATP opposite T and
C (Table 3) were comparable to those of 8-OH-dGTP reported with
exonuclease-free KF(19) . Therefore, 2-OH-dATP appears to be
incorporated as effectively as 8-OH-dGTP by DNA polymerases.
In
aqueous solution, the major tautomers of the 9-methyl and ribosyl
derivatives of 2-OH-Ade are in the N(1)H, 2-keto form(17) . The
keto and enol forms are calculated to be about 90% and about 10%,
respectively, of the total in water(17) . The postulated base
pairs of 2-OH-Ade with T and C are shown in Fig. 4(A and B). With T, the enol tautomer of 2-OH-Ade can form a
pair in a Watson-Crick manner, although the keto tautomer can also pair
in a wobble alignment. In this putative Watson-Crick pair, the two
bases form three hydrogen bonds as GC. With C, both tautomers of
2-OH-Ade can form wobble pairs. The kinetic data obtained in this study
indicated that the V
value of T
2-OH-Ade
with pol
was larger than that of T
A, although the K
value of T
2-OH-Ade was larger (Table 3). This fact may be explained by the small ratio of the
enol tautomer in the solution used in this study, and by rapid
phosphodiester bond formation due to the more stable hydrogen bonding
of T
2-OH-Ade (enol) than T
A.
Figure 4:
Postulated base pairs involving 2-OH-Ade. A, 2-OH-AdeT pair; B, 2-OH-Ade
C pair; C: 2-OH-Ade
A pair.
We recently found that KF
and pol inserted dAMP as well as dTMP opposite 2-OH-Ade in in
vitro DNA synthesis(18) . On the other hand, the two DNA
polymerases did not insert 2-OH-dATP opposite A in template DNAs, and
pol
incorporated 2-OH-dATP opposite C (Figs. 2 and 3). This
discrepancy may depend on the different sequence contexts used in both
studies (see ``Results''). However, we did not observe the
incorporation of 2-OH-dATP opposite A and G in the template that has
the same flanking sequences near the target position (Table 2).
The reasons for the different tendencies remain to be resolved. One
possibility is that 2-OH-Ade can form a base pair with A when the
modified base is in the enol form (Fig. 4C). Sepiol et al. reported that the equilibrium between the keto and enol
forms of 2-OH-Ade shifts in the direction of the enol form with a
decrease in the solvent polarity(17) . It is known that a base
moiety in DNA is surrounded by neighboring bases that are stabilized by
stacking, and is in a more hydrophobic environment than the monomer.
Therefore, the structure of 2-OH-Ade in DNA may shift more to the
2-enol form than in the monomer. Furthermore, the DNA molecule is
surrounded by a DNA polymerase when DNA synthesis occurs. The polarity
of the active site of a DNA polymerase may be less polar than water.
Thus, the structure of 2-OH-Ade in a template DNA to which a DNA
polymerase binds may significantly shift to the enol form, as suggested
by Switzer et al.(11) , and may pair with A (Fig. 4C). On the other hand, the ratio of the enol
form in 2-OH-dATP is probably similar to that in the 9-methyl and
ribosyl derivatives of 2-OH-Ade. If the equilibrium shifts from the
keto to the enol form slowly, then 2-OH-dATP exists almost in the keto
form, even near the active site of a DNA polymerase, and could
dissociate from the position opposite A in a template. In contrast, the
facts that 2-OH-dAMP was incorporated opposite C (Fig. 3), and
that dCTP was not inserted opposite 2-OH-Ade in the
template(18) , may be explained if C pairs with the keto form
of 2-OH-Ade (Fig. 4B).
In the case of 8-OH-dGTP, the MutT protein and its homologues prevent the mutagenic nucleotide from being incorporated into DNA, by hydrolyzing 8-OH-dGTP to 8-OH-dGMP(9, 20) . A MutT-like activity that destroys 2-OH-dATP may be present in bacterial and mammalian cells.
In this
report, we have shown that 2-OH-Ade is formed by oxygen radicals, and
that KF and a replicative polymerase, pol , incorporated
2-OH-dATP. The latter enzyme inserted the nucleotide opposite C, in
addition to T, in template DNAs.