Download 6-Methoxyadenine Residue Forms a Watson

Article No. jmbi.1999.3303 available online at http://www.idealibrary.com on
J. Mol. Biol. (1999) 294, 1215±1222
Crystallographic Studies on Damaged DNAs. I. An
N 6-Methoxyadenine Residue Forms a Watson-Crick
Pair with a Cytosine Residue in a B-DNA Duplex
Toshiyuki Chatake1, Akira Ono2, Yoshihito Ueno3, Akira Matsuda3
and Akio TakeÂnaka1*
1
Graduate School of Bioscience
and Biotechnology, Tokyo
Institute of Technology
Nagatsuda, Midori-ku
Yokohama, 226-8501, Japan
2
Graduate School of Science
Tokyo Metropolitan University
Hachioji, Minamiosawa, Tokyo,
192-0364, Japan
3
Graduate School of
Pharmaceutical Sciences
Hokkaido University, Nishi 6,
Kita 12, Kita-ku, Sapporo,
060-0812, Japan
Oxyamines such as hydroxylamine and methoxylamine disturb DNA
replication and act as potent mutagens, causing nucleotide transition
from one purine to another or one pyrimidine to another. In order to
investigate mismatch base-pairing in DNA damaged with oxyamines, a
dodecamer with the sequence d(CGCGmo6AATCCGCG), where mo6 A is
20 -deoxy-N6-methoxyadenosine, was synthesized and its crystal structure
determined. No signi®cant conformational changes are found between
the present dodecamer and the original undamaged B-form dodecamer.
Electron density maps clearly show that the mo6A residue forms a basepair with a 20 -deoxycytidine residue through hydrogen bonds similar to a
Watson-Crick G C base-pair. For these hydrogen bonds to be made,
N6-methoxyadenine must chemically take the imino form. The methoxylation thus enables the adenine base to mimic a guanine base. As a result,
misincorporation of 20 -deoxycytidine instead of thymidine, or 20 -deoxyadenosine instead of 20 -deoxyguanosine, can occur in DNA replication.
# 1999 Academic Press
6
*Corresponding author
Keywords: N -methoxyadenine; mutagenesis; X-ray structure; mismatch;
damaged DNA
Introduction
In all organisms, the very high accuracy of DNA
replication is achieved by using the Watson-Crick
pairs between adenine and thymine residues and
between guanine and cytosine residues as an
absolute principle. However, oxyamines such as
hydroxylamine and methoxylamine disturb this
rule and act as potent mutagens, causing nucleotide transitions from one purine to another purine,
or from one pyrimidine to another pyrimidine
(Singer & Kusmierek, 1982). These chemicals predominantly attack and modify the exocyclic amino
groups of nucleic acid bases (Kochetkov &
Budowsky, 1969). Substitutions at the N4 amino
group of cytosine and at the N6 amino group of
adenine have been identi®ed and considered to be
Abbreviations used: dATP, 20 -deoxyadenosine 50 triphosphate; TTP, thymidine 50 -triphosphate; dGTP, 20 deoxyguanosine 50 -triphosphate; dCTP,
20 -deoxycytidine 50 -triphosphate; mo6A, 20 -deoxyN6-methoxyadenosine; mo6dATP, 20 -deoxy-N6methoxyadenosine 50 -triphosphate.
E-mail address of the corresponding author:
[email protected]
0022-2836/99/501215±8 $30.00/0
the origin of the mutation (Budowsky et al., 1975).
Matsuda and co-workers (Nishio et al., 1992) found
that when adenine residues of a template DNA
strand were methoxylated at N6, the 20 -deoxycytidine 50 -triphosphate (dCTP) as well as the complementary thymidine 50 -triphosphate (TTP) were
incorporated into the newly synthesized DNA
strand at the opposite site to 20 -deoxy-N6-methoxyadenosine (hereafter 20 -deoxy-N6-methoxyadenosine is designated as mo6A). On the other hand,
when 20 -deoxyadenosine 50 -triphosphate (dATP) is
methoxylated (mo6dATP), mo6dATP is incorporated at the opposite sites to both 20 -deoxycytidine
and thymidine residues of a template strand
(Singer & Spengler, 1982; Abdul-Maish & Bessman,
1986; Hill et al., 1998). It is interesting to reveal
how such a non-Watson-Crick nucleotide is incorporated, despite the fact that the DNA polymerase
accepts only Watson-Crick base-pairs (Kiefer et al.,
1998).
Crystallographic studies have been reported on
the structural properties of methoxylated adenine.
Two crystal structures of N6-methoxyadenine
derivatives, N9-benzyl-N6-methoxyadenine (Fujii
et al., 1990) and N6-methoxy-20 ,30 ,50 -tri-O-methyl# 1999 Academic Press
1216
adenosine (Birnbaum et al., 1984), indicate that the
N6-methoxylated base prefers the imino form. This
fact is consistent with the results from proton
nuclear magnetic resonance (Stolarski et al., 1984,
1987), and ultraviolet and infrared spectroscopy
studies (Fujii et al., 1987). In the crystals, the methoxy groups take a syn conformation around the
N6-C6 bond to the N1 atom, so that the two purine
moieties are associated through three hydrogen
bonds including a C-H N interaction. From
these results, Birnbaum et al. (1984) proposed that
mo6A with a syn conformation pairs with a 20 deoxycytidine through two hydrogen bonds,
between N1(mo6A) and N3(C) and between the
methoxy oxygen atom and N4(C). The geometry of
the base-pair, however, will be largely distorted
from that of the regular Watson-Crick pairing.
Such a distortion might prevent incorporation of
the modi®ed adenine base into the active site of
DNA polymerase, because the incoming nucleotides are strictly selected for their ability to form a
Watson-Crick base pairing (Kiefer et al., 1998).
In order to investigate structurally the mismatch
base-pairing, X-ray analysis of DNA oligomers containing mo6A should be an ideal approach. Unfortunately, this has not been possible so far: no X-ray
structures of DNA oligomers containing N6-methoxyadenine or N6-hydroxyadenine have been
reported. Only the crystal structure of a DNA hexamer containing 20 -deoxy-N4-methoxycytidine with
the sequence d(CGCGXG) (X is 20 -deoxy-N4-methoxycytidine) was reported (Meervelt et al., 1990).
The N4-methoxy group takes the imino form, consistent with solution studies (Brown et al., 1968;
Morozov et al., 1982). However, the hexamer adopts
a Z-form and the methoxy group is in a syn conformation around the N4-C4 bond, forming a wobble
base-pair with the guanine moiety on the opposite
strand. Similar structures have also been found in
other hexamers containing 20 -deoxy-b-D-ribo
furanosyl-(6H,8H-3,4-dihydro-pyrimido[4,5-c][1,2]
oxazin-7-one) as X (Moore et al., 1995). These structural features are unacceptable to the DNA polymerase (Kiefer et al., 1998). In addition, only the
wobbled base-pairing has been found between cytosine bases and adenine bases in the protonated
form (Schuerman et al., 1998; Hunter et al., 1986).
To understand the structural basis of mutation
mechanism, it is necessary to clarify whether the
modi®ed adenine moiety can form a Watson-Crick
pair with a cytosine base. Therefore, DNA dodecamer containing 20 -deoxy-N6-methoxyadenosine,
with the sequence d(CGCGmo6AATCCGCG)
(hereafter Dmo6A C), has been synthesized and its
crystal structure determined. This dodecamer was
designed so that mo6A interacts with C upon
duplex formation, since it is known that the original sequence d(CGCGAATTCGCG) (referred to
hereafter as the original dodecamer) prefers to take
a B-form conformation (Dickerson & Drew, 1981;
Shui et al., 1998). The X-ray structure reported here
shows that the methoxy group has no signi®cant
effects on the overall DNA conformation. Electron
Watson-Crick type N6-Methoxyadenine Cytosine Pair
density maps clearly show that the mo6A residue
forms a Watson-Crick-like base-pair with the cytosine residue on the opposite strand. This is the ®rst
example of such a pairing between N6-methoxyadenine and a cytosine base. We describe below
the details of the novel hydrogen-bonding scheme
thus revealed in B-form duplex DNA.
Results and Discussion
Overall structure
The crystal structure of Dmo6A C is isomorphous to that of the original dodecamer (Dickerson
& Drew, 1981; Shui et al., 1998). The two Dmo6A C
strands form a duplex, as expected (Figure 1). The
overall structures of the two dodecamers with and
without methoxy groups are very similar to each
other, their root-mean-square displacement being
Ê on superimposition of non-hydrogen atoms.
0.31 A
All torsion angles and local helical parameters calculated by the program NUPARM (Bansal et al.,
1995) are given in the Supplementary Material
(Tables 1 and 2). Figure 2 shows plots of some
representative helical parameters along the nucleotide sequence. These parameters ¯uctuate around
average values close to those of typical B-form
DNA; their variations along the two sequences are
almost the same. Thus, the methoxylation of adenosine residues does not affect the DNA conformation signi®cantly.
Crystal packing
Two duplexes related by a crystallographic 21
symmetry along the c-axis in the crystal form columns in a head-to-tail fashion. The guanine residue
at one end of one duplex forms a base-pair
with the terminal guanine base at the other end of
the symmetry-related duplex, through two
Figure 1. A stereo view of the ``damaged'' DNA
dodecamer containing 20 -deoxy-N6-methoxyadenosine.
The diagram was drawn with the program MOLSCRIPT
(Kraulis, 1991). The mo6A residues are shown in red
and the cytosine bases paired with mo6A are in green.
1217
Watson-Crick type N6-Methoxyadenine Cytosine Pair
Figure 3. The guanine-guanine hydrogen bonds
between two symmetry-related duplexes (see the text).
The nucleotides are numbered from the 50 end independently in the two strands, a and b.
Figure 2. A comparison of the helical parameters of
Dmo6A C (circles) with those of the original dodecamer
(boxes) (Shui et al., 1998). The top, middle and bottom
diagrams, represent the helical rises, the helical twist
angles and the displacements of the two duplexes,
respectively, plotted along the nucleotide sequence to
show their ¯uctuations. These parameters were calculated by the program NUPARM (Bansal et al., 1995).
In the right, shaded column, their average values are
indicated, together with the typical values for standard
A and B-DNA.
N2-H N3 hydrogen bonds (see Figure 3). As
reported in a previous paper (Chatake et al.,
1999b), during data collection, a phase transition of
the Dmo6A C crystal with changing humidity at
room temperature was found. Water molecules
being incorporated into the crystal cause this
change. As a result, the contact angle between two
neighboring duplexes is changed from 132 to 144 .
The present crystal structure has been determined
at 110 K. The corresponding angle in the present
crystal is 146 , and the unit cell is slightly shrunk,
though the variation is within experimental error.
An octahedrally hydrated magnesium cation is
located in the major groove of one duplex and contacts the ribose-phosphate backbone of another
duplex, the position being almost the same as that
found in the original dodecamer. The cation links
the two duplexes laterally through hydrogen
bonds, as shown in Figure 4. Three of the Mg2‡coordinating water molecules form hydrogen
bonds with guanine bases, and the other three are
hydrogen bonded to the oxygen atoms of the phosphate groups of another duplex, related by a 21
symmetry along the b-axis (Figure 4). Other water
molecules (with the exception of those around the
methoxy groups) are also located at similar positions to those of the original dodecamer, and form
spines in the grooves of the duplex. Some of the
water positions in the minor groove might be partially occupied by sodium cations, as proposed by
Shui et al. (1998). The secondary spine in the minor
groove rides ``piggyback'' on the primary spine.
Tautomerism of mo6A and hydrogenbonding scheme
Final 2jFoj ÿ jFcj maps of the individual basepairs are shown in Figure 5. At the ®fth and eighth
base-pairs, the densities are well resolved and
clearly reveal the positions of the two mo6A residues. The most interesting feature is that both
methoxylated adenines pair with cytosine bases
in a geometry very similar to the Watson-Crick
adenine thymine pair. The average temperature
Ê 2 and
factors of these two base-pairs (12.6 A
Ê 2) are comparable to those of the other base13.4 A
pairs, indicating that the two base-pairs,
mo6Aa5 Cb8 and mo6Ab5 Ca8 (see the nucleotide
numbering in Figure 5) each occupy a single con®guration. Their closest atomic distances and the
atomic valence angles are given in Table 1; those of
the other hydrogen bonds are given in the
Supplementary Material (Table 3). They are in an
acceptable range for the N-H N hydrogen
bond. To form base-pairs through these hydrogen
1218
Watson-Crick type N6-Methoxyadenine Cytosine Pair
mation may be induced to stabilize the base-pairing with cytosine.
Biological significance
Figure 4. The octahedrally hydrated magnesium cation which links two neighbouring duplexes through
hydrogen bonds. Ow indicates water molecules.
Ê.
Distances are given in A
bonds, the chemical structure of the methoxylated
adenine base must be the imino form, as shown in
Figure 6, since it is known that the cytosine base
exists only in the amino form (Pieber et al., 1973).
This is consistent with the structures found in N6methoxyadenine derivatives (Birnbaum et al., 1984;
Fujii et al., 1990), which take the imino form.
Although many types of base-pairs have been
reported, only the wobble base-pairing has been
found between cytosine and adenine bases in the
protonated form (Schuerman et al., 1998; Hunter
et al., 1986). Therefore, the present structure is the
®rst example of a Watson-Crick pair between N6methoxyadenine and cytosine. It clearly reveals the
structural basis of the mutation mechanism associated with the damaged DNA.
The second striking feature is that both methoxy
groups adopt an anti conformation to the N1 atom
around the C6-N6 bond. This conformation is quite
different from those found previously in N6-methoxyadenine derivatives (Birnbaum et al., 1984; Fujii
et al., 1990). In the latter crystals, the derivatives
are in a syn conformation, forming a hydrogenbonded ribbon involving C-H O and C-H N
interactions. Although the tautomers are in the
same imino form as here, the conformations of the
methoxy groups are different. To change the conformation from syn to anti or vice versa, the adenine
moiety must pass through the amino form by tautomerization. Since it is thought that N6-methoxyadenine derivatives are in equilibrium between the
amino and the imino forms in solution (Stolarski
et al., 1984), the imino form with the anti confor-
During replication of DNA, the polymerase
accepts only Watson-Crick type base pairs (Kiefer
et al., 1998). To form such a pair with a cytosine
residue, the adenine moiety must be in the imino
form. The unmodi®ed adenine residue never
adopts such a tautomer (Wolfenden, 1969). On the
other hand, in the structures of N6-methoxyadenine
derivatives (Fujii et al., 1990; Birnbaum et al., 1984),
including the present dodecamer, the adenine moieties are all in the imino form. The donor/acceptor
sites for hydrogen bonds are changed, so that the
methoxylated adenine can mimic guanine in
hydrogen bond formation. This mimicry makes it
possible to form a Watson-Crick type base-pair
with a cytosine residue. In the imino form of
mo6A, there are two possible conformations for the
methoxy group around the C6-N6 bond. Although
Birnbaum et al. (1984) proposed a pairing between
mo6A in a syn conformation and a cytosine residue, the corresponding geometry is largely distorted from that of regular Watson-Crick pairs.
Such a distortion might prevent incorporation of
the modi®ed adenine residue into the active site of
DNA polymerase (Kiefer et al., 1998). In contrast,
mo6A in the present structure is in an anti conformation. When the mo6A C pairs found in the
present investigation are superimposed on the
canonical A T pairs of the original dodecamer, the
base positions are almost the same. This structural
isomorphism will almost certainly favor recognition by the polymerase. In addition, the polymerase has an open, solvent accessible space in the
major groove of the bound DNA, so that the methoxy groups on the damaged DNA will not interfere
with its binding to the polymerase. Through these
structural characteristics, an mo6A residue on the
template strand makes it possible for dCTP to be
incorporated at the opposite site in the newly synthesized DNA strand (Nishio et al., 1992). In the
same way, mo6dATP can be incorporated at the
site opposite to a cytosine residue on the template
strand (Singer & Spengler, 1982; Abdul-Maish &
Bessman, 1986; Hill et al., 1998).
The equilibrium between the amino and the
imino forms of N6-methoxyadenine changes with
the polarity of the solution (Stolarski et al., 1984).
In addition, when a cytosine derivative is added to
a solution containing an mo6A derivative, the
imino form is increased, whereas when a uracil
derivative is added, the amino form is increased
(Stolarski et al., 1987). Therefore, it is expected that
an mo6A residue in the amino form will make a
Watson-Crick base-pair with a thymine residue.
The crystal structure of a DNA dodecamer containing such base-pairs will be discussed in the following paper (Chatake et al., 1999a).
Watson-Crick type N6-Methoxyadenine Cytosine Pair
1219
Figure 5. Final re®ned 2jFoj ÿ jFcj electron density maps superimposed on all base-pairs. The maps were contoured
at the 1s level by the program O (Jones et al., 1991). The nucleotides are numbered from the 50 end independently in
the two strands, a and b.
Experimental Procedures
Synthesis and crystallization
A DNA dodecamer of Dmo6A C was synthesized and
puri®ed by the reported method (Nishio et al., 1992).
Crystallization was carried out at 4 C by the hanging
drop vapor diffusion method in a 4 ml droplet containing
equal volumes of 1.5 mM DNA solution in water and
reservoir solution. Suitable crystals of Dmo6A C were
obtained from 35 % (v/v) 2-methyl-2,4-pentanediol,
18 mM magnesium acetate, 6 mM spermine tetrahydrochloride and 10 mM sodium cacodylate (pH 7.0).
1220
Watson-Crick type N6-Methoxyadenine Cytosine Pair
Ê ) and angles (deg.) of mo6A C base-pairs
Table 1. Hydrogen bond distances (A
mo6Aa5 Cb8
Ca8 mo6Ab5
N1 N3
C2-N1 N3
N1 N3-C2
N3 N1
C2-N3 N1
N3 N1-C2
2.76
123
112
2.80
109
123
N6 N4
C6-N1 N3
N1 N3-C4
N4 N6
C4-N3 N1
N3 N1-C6
Data collection
The crystals were ¯ash-frozen in a stream of nitrogen
at 110 K. X-ray data were collected at 110 K on the
Sakabe-Weissenberg camera (Sakabe, 1991) with
synchrotron radiation at the Photon Factory (BL-6B) in
Tsukuba. To compensate the blind region, another
crystal with a different orientation was used and the
X-ray diffraction was collected at 110 K on a Rigaku RAXIS IIc with CuKa radiation generated at 50 kV and 80
mA. Diffraction patterns recorded on imaging plates
were processed by the program DENZO (Otwinowski &
Minor, 1997) and intensity data were merged by the programs SCALA and AGROVATA in CCP4 (Collaborative
Computational Project Number 4, 1994). A total of 8017
independent re¯ections with an Rmerge of 6.0 % were
obtained from 32,780 observed re¯ections. The completeÊ resolution
ness of the data was 87.1 % in the 100-1.6 A
Ê resolution shell.
range and 72.1 % for the outer 1.69-1.6 A
Ê , b ˆ 39.9 A
Ê,
The cell dimensions are a ˆ 25.1 A
Ê , the space group being P212121. Statistics of
c ˆ 65.8 A
data collection and crystal data are summarized in
Table 2.
Structure determination
Initial phases were determined by the molecular replacement method using the structure of the original dodecamer (Dickerson & Drew, 1981; Shui et al., 1998) with
the program AMoRe (Navaza, 1994). The molecular
structure was constructed and modi®ed on a graphic
workstation by inspecting jFoj ÿ jFcj omit maps at every
nucleotide residue with the programs O (Jones et al.,
1991) and QUANTA (distributed by Molecular Simulations, Inc.). The jFoj ÿ jFcj omit maps clearly showed
that the mo6A residues form base-pairs with cytosine
residues, the methoxy groups taking an anti conformation to the N1 atom around the N6-C6 bond. From the
hydrogen-bonding scheme, the mo6A residues were
assumed to take the imino form. The stereochemical
parameters employed for structural re®nement with the
program X-PLOR (BruÈnger, 1992b) were those in the dictionary ®le dna-rna.param. For the present re®nements,
this ®le was modi®ed to include the 20 -deoxy-N6-methox-
2.87
112
128
3.10
131
113
122
114
C4-N4 N6
N4 N6-C6
112
120
O6-N6 N4
125
N4 N6-O6
127
yadenosine residue with the imino form. Its structural
parameters were derived from the crystal structures of
mo6A derivatives (Fujii et al., 1990; Birnbaum et al.,
1984). The crystal structure was re®ned through a combination of simulated annealing and positional re®nements, followed by interpretation of omit maps at every
nucleotide residue. During the above re®nement, no
hydrogen bonding restraints were applied between
paired nucleotides. A total of 173 peaks were assigned as
water molecules. One magnesium cation coordinated
octahedrally by six water molecules was found. The ®nal
Ê resolution data
R-factor was 19.6 % for 5.0 1.6 A
(Rfree ˆ 23.1 % for 10 % of the observed data). Statistics of
the structure determination are summarized in Table 2.
Data Bank accession codes
The atomic coordinates have been deposited in the
Nucleic Acid Database (NDB) (entry code BD0009).
Table 2. Crystal data, data collection and structure
determination
Space group
Ê)
Unit cell (A
Asymmetric unit (duplexes)
Ê)
Resolution (A
Measured reflections
Unique reflections
Completeness (%)
In the Outershell (%)
Rmerge a (%)
Structure refinement
Resolution range
Used reflections
Final model
Number of non-hydrogen
atoms
Number of water molecules
Number of magnesium
atoms
Ê 2)
Average B-factors (A
DNA
Water molecules
Magnesium atom
R-factorb (%)
Rfree c (%)
r.m.s. deviation
Ê)
Bond lengths (A
Bond angles (deg.)
Improper angles (deg.)
Ê)
Average coordinates errord (A
a
P212121
a ˆ 25.1, b ˆ 39.9, c ˆ 65.8
4
100 1.6
32,780
8017
87.1
Ê)
72.1(1.69 1.6 A
6.0
5.0 1.6
7676
488
173
1
17.3
37.9
15.8
19.6
23.1
0.009
1.4
1.6
0.20
Rmerge ˆ 100 hkljjIhklj ÿ hIhklij/hklhIhkli.
R-factor ˆ 100 jjFoj ÿ jFcjj/jFoj, where jFoj and jFcj
are the observed and calculated structure factor amplitudes,
respectively.
c
Calculated using a random set containing 10 % of observations that were omitted during re®nement (BruÈnger, 1992a).
d
Estimated from a Luzzati plot (Luzzati, 1952).
b
Figure 6. Chemical structure of mo6A with possible
hydrogen bonds.
C6-N6 N4
N6 N4-C4
Watson-Crick type N6-Methoxyadenine Cytosine Pair
Acknowledgments
We thank N. Sakabe and N. Watanabe for facilities
and help during data collection at the Photon Factory
(Tsukuba), and T. Simonson for proofreading of the
manuscript. This work was supported in part by a grant
for the RFTF(97L00503) (Research For The Future) from
the Japanese Society for the Promotion of Science, by
Grants-in-Aid for Scienti®c Research on Priority Area
(Nos. 08124203 and 07250205) from the Ministry of Education, Science, Sport and Culture of Japan, and by the
Sakabe project of TARA (Tsukuba Advanced Research
Alliance), University of Tsukuba.
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Edited by I. Tinoco
(Received 19 April 1999; received in revised form 6
October 1999; accepted 12 October 1999)
http://www.academicpress.com/jmb
Supplementary Material comprising three Tables is
available from JMB Online