Implications for Damage Recognition during Dpo4Mediated Mutagenic Bypass of m1G and m3C Lesions
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Citation
Rechkoblit, Olga, James C. Delaney, John M. Essigmann, and
Dinshaw J. Patel. “Implications for Damage Recognition During
Dpo4-Mediated Mutagenic Bypass of m1G and m3C Lesions.”
Structure 19, no. 6 (June 2011): 821–832. © 2011 Elsevier Ltd.
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http://dx.doi.org/10.1016/j.str.2011.03.020
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Detailed Terms
Structure
Article
Implications for Damage Recognition
during Dpo4-Mediated Mutagenic Bypass
of m1G and m3C Lesions
Olga Rechkoblit,1,4 James C. Delaney,2,3 John M. Essigmann,2 and Dinshaw J. Patel1,*
1Structural
Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
of Chemistry and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3Visterra Inc., Cambridge, MA 02139, USA
4Present address: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY 10029, USA
*Correspondence: pateld@mskcc.org
DOI 10.1016/j.str.2011.03.020
2Department
SUMMARY
DNA is susceptible to alkylation damage by a number
of environmental agents that modify the WatsonCrick edge of the bases. Such lesions, if not repaired,
may be bypassed by Y-family DNA polymerases. The
bypass polymerase Dpo4 is strongly inhibited by
1-methylguanine (m1G) and 3-methylcytosine
(m3C), with nucleotide incorporation opposite these
lesions being predominantly mutagenic. Further,
extension after insertion of both correct and incorrect
bases, introduces additional base substitution and
deletion errors. Crystal structures of the Dpo4 ternary
extension complexes with correct and mismatched
30 -terminal primer bases opposite the lesions reveal
that both m1G and m3C remain positioned within
the DNA template/primer helix. However, both
correct and incorrect pairing partners exhibit
pronounced primer terminal nucleotide distortion,
being primarily evicted from the DNA helix when
opposite m1G or misaligned when pairing with m3C.
Our studies provide insights into mechanisms related
to hindered and mutagenic bypass of methylated
lesions and models associated with damage recognition by repair demethylases.
INTRODUCTION
Alkylation damage to DNA is induced by naturally occurring
endogenous agents (De Bont and van Larebeke, 2004; Sedgwick, 1997), as well as by many environmental carcinogens
(Hecht, 1999; Singer and Grunberger, 1983) and by cancer
chemotherapeutics (Allan and Travis, 2005; Park et al., 2010).
Secondary cancers that arise as a consequence of chemotherapy with alkylating agents are not unusual and are associated with mutation accumulation, genome instability and defects
in DNA repair (Allan and Travis, 2005). Among the diverse lesions
produced by the SN2 type alkylating agents are methylation at
the N1-position of adenine (m1A) and guanine (m1G), and at
the N3-positions of cytosine (m3C) and thymine (m3T) (Figure 1A)
(Sedgwick, 2004; Shrivastav et al., 2010).
The addition of a methyl group to the endocyclic N-atoms that
are normally involved in Watson-Crick base pairing is cytotoxic
and blocks DNA replication in Escherichia coli in the absence
of the AlkB repair protein (Delaney and Essigmann, 2004). This
result suggests that the polymerase responsible for replicating
genomic DNA, presumably the B-family Pol III, in E. coli, would
be drastically impeded by the m1A, m1G, m3C, and m3T lesions.
In vitro data indicate that the A-family gap-filling E. coli Pol I Klenow fragment is severally hindered by the m1A (Larson et al.,
1985), m3C (Boiteux and Laval, 1982; Saffhill, 1984), and m3T
(Huff and Topal, 1987) lesions. Activation of the SOS-response
in E. coli induces production of Pol IV and Pol V, the low-fidelity
Y-family translesion synthesis (TLS) polymerases (Yang and
Woodgate, 2007), which increases bypass of the m1A, m3C,
m1G, and m3T lesions in vivo by 3–4-fold (Delaney and Essigmann, 2004). The resulting progeny contain a staggering fraction
of mutations: 70% in the case of the m3C- and m1G-modified
templates and 53% in the case of the m3T; only m1A is weakly
mutagenic generating 1% of errors (Delaney and Essigmann,
2004). In eukaryotes, as in E. coli, Y-family polymerases temporarily replace stalled high-fidelity polymerases with damagebypass polymerases (McCulloch and Kunkel, 2008; Waters
et al., 2009; Yang and Woodgate, 2007). Currently, structurefunction studies that address TLS polymerase-catalyzed bypass
of blocking m1A, m3C, m1G, and m3T lesions are lacking.
In contrast to high-fidelity B- and A-family polymerases that
produce tight-fitting, solvent-excluding reaction-ready active
sites on binding of a complementary dNTP (Johnson and Beese,
2004; Steitz and Yin, 2004; Swan et al., 2009a), Y-family polymerases have more spacious and solvent-accessible active
sites (Biertumpfel et al., 2010; Silverstein et al., 2010; Yang and
Woodgate, 2007). Furthermore, Y-family polymerases do not
have an exonuclease domain. Additionally, they do not check
the minor groove edge of the template/primer to proofread
mismatches and they select for the correct dNTP guided
predominantly by base pairing complementarity (Yang and
Woodgate, 2007). These structural and functional features
enable Y-family polymerases to bypass a variety of DNA lesions
and at the same time cause a higher error rate on undamaged
DNA templates (Broyde et al., 2008; Waters et al., 2009; Yang
and Woodgate, 2007).
Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved 821
Structure
Bypass of Methylation Damage Lesions by Dpo4
A
NH2
+ CH 3
N
1N
N
C
O
NH2
N
CH 3
N
1N
m1A
dR
N
Finger
Palm
m1G
B
A
NH2
N
dR
O
NH2
+ CH3
3N
H 3C
3N
m3C
N
N
O
dR
CH3
m3T
O
Little
finger
Thumb
dR
dR = 2′–deoxyribose
Template
Primer
m1G- and m3C-modified Dpo4 extension ternary complexes
B
(0)
1 2 3 4 5
(–1)
6
(–2)(–3)
7 8 9 10 11 12 13 14 15 16 17 18 19
Template
•
Primer
•
14
= m1G or m3C
= C, A, G, or T
13 12 11 10 9 8 7 6 5 4 3 2
E. coli AlkB is an iron(II)-and 2-oxoglutarate-dependent repair
protein that directly reverses alkylation damage on the endocyclic N-atoms of DNA and RNA bases by oxidative demethylation (Falnes et al., 2002; Trewick et al., 2002). AlkB proficiently
removes methyl groups from m1A and m3C adducts (Sedgwick
et al., 2007; Shrivastav et al., 2010) predominantly in singlestranded DNA, but it is markedly less efficient on the more relatively minor m1G and m3T lesions (Delaney and Essigmann,
2004; Falnes et al., 2002; Trewick et al., 2002; Yu and Hunt,
2009). The human genome encodes at least nine AlkB family
members, ABH1 through ABH8 (Aravind and Koonin, 2001;
Sedgwick et al., 2007) and the fat mass- and obesity-associated FTO protein (Gerken et al., 2007). ABH2 is recognized
as a primary demethylase that protects genomic DNA, because
mice lacking ABH2 accumulate significant levels of m1A in their
genome even in the absence of any exogenous methylating
agent (Ringvoll et al., 2006). Consistently, in vitro, ABH2 works
preferentially on double-stranded DNA and repairs m1A, m3C,
and m3T (Aas et al., 2003; Duncan et al., 2002; Falnes, 2004;
Ringvoll et al., 2006); m1G-containing DNA was not evaluated.
ABH1 (Westbye et al., 2008), ABH3 (Aas et al., 2003; Duncan
et al., 2002; Falnes, 2004; Ringvoll et al., 2006), and FTO
(Gerken et al., 2007; Jia et al., 2008) are most efficient on
ssDNA and ssRNA; however, their substrate specificities are
different. The exact cellular roles of ABH1 and ABH3, as well
as ABH4–7 remain elusive. Surprisingly, FTO is a key factor in
energy homeostasis regulation (Fischer et al., 2009), and
ABH8 is involved in methylation of tRNA and participates in
regulation of the DNA-damage response pathway (Fu et al.,
2010a, 2010b).
The crystal structures of AlkB with short ssDNA (Yu et al.,
2006) and AlkB and ABH2 (Yang et al., 2008) with dsDNA,
where both ss- and dsDNAs contained the m1A lesion, as
well as ABH3 in the absence of DNA or RNA (Sundheim
et al., 2006) and FTO with the m3C nucleobase (Han et al.,
2010) revealed strikingly similar overall folds of the catalytic
Figure 1. Lesions, Sequences, and Overall
Structure of the Dpo4 Extension Ternary
Complexes
(A) Chemical structures for the major m1A and m3C
and the minor m1G and m3T alkylation damage
lesions (Sedgwick, 2004; Shrivastav et al., 2010).
(B) Schematic of the m1G- and m3C-modified 19mer templates with the 13-mer primers, ending
with a 20 ,30 -dideoxy-C, A, G, or T, and dGTP in the
extension ternary complex with Dpo4; the
templates have a single unpaired residue at the 30 end. The insertion position at the Dpo4 active site
is denoted by (0), and the post-insertion position is
denoted by (–1).
(C) Overall structure of the m1G,C complex. The
first Ca2+, cation A, is coordinated by the catalytic
D7, D105, and E106 residues, the second Ca2+,
cation B, is chelated by the phosphate groups of
the incoming dGTP. The unlabeled third ion is
found in Dpo4 ternary complexes (Bauer et al.,
2007; Eoff et al., 2007; Ling et al., 2001, 2003;
Rechkoblit et al., 2006, 2010; Zhang et al., 2009)
and other Y-family polymerases (Lone et al., 2007;
Nair et al., 2004; Swan et al., 2009b).
domains. These proteins flip damaged bases and insert them
into the active site for repair (Han et al., 2010; Sedgwick
et al., 2007; Sundheim et al., 2006; Yang et al., 2008; Yu
et al., 2006). Thus far only ABH2 has been identified as a dsDNA
repair protein; it interacts with both damaged and undamaged
strands and employs an aromatic residue to intercalate into the
duplex DNA and fill the gap resulting from the base flipping
(Han et al., 2010; Yang et al., 2008). It is currently unclear
how ABH2 identifies damaged bases opposite their cognate
partners in the context of the vast amount of undamaged
dsDNA. Moreover, no data are available on lesion recognition
opposite incorrect base partners that were mistakenly incorporated by DNA polymerases during replication. The structures of
the cognate m1A,T and m3C,G alignments within dsDNA
duplexes have provided valuable information into a possible
DNA scanning mechanism employed by ABH2 (Lu et al.,
2010), but much remains to be elucidated.
The objectives of this work were to explore structural and
biochemical features of Dpo4-catalyzed bypass of the m1G
and m3C lesions. We demonstrate that this TLS polymerase,
that is able to efficiently and in many cases accurately bypass
a variety of DNA lesions ranging in size and chemical functionality (Bauer et al., 2007; Eoff et al., 2007; Ling et al., 2003;
Rechkoblit et al., 2006, 2010; Zhang et al., 2009), is strongly
inhibited by m1G and m3C lesions. Furthermore, the extension
products, which are nevertheless generated during bypass, are
predominately mutagenic. The crystal structures of eight ternary
Dpo4 complexes, with either correct or incorrect terminal
30 -partner bases opposite m1G and m3C lesions and cognate
dNTP paired with a template base 50 to the lesion site, reveal
the basis of the observed blockage and mutagenicity. Moreover,
the observed alignments of m1G- and m3C- lesions with C, T, A,
and G partner bases, which reveal pronounced primer terminal
nucleotide distortion, have important implications for the efficiency of recognition by ABH2 and, thus, the cytotoxicity and
mutagenicity of these lesions.
822 Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Bypass of Methylation Damage Lesions by Dpo4
Table 1. Data Collection and Refinement Statistics
m1G,C
m1G,T
m1G,A
m1G,G
m3C,C
m3C,T
m3C,A
m3C,G
P1
P1
P21212
P21
P21
P21
P21
P21
a, b, c (Å)
52.2, 62.1,
91.5
52.9, 61.6,
92.7
93.2, 110.4,
51.9
52.4, 110.4,
102.3
52.9, 109.3,
100.7
52.9, 109.9,
100.3
52.9, 109.3,
100.8
52.3, 110.0,
101.5
a, b, g ( )
99.0, 103.8,
93.7
98.5, 103.8,
93.3
90.0, 90.0,
90.0
90.0, 101.3,
90.0
90.0, 101.1,
90.0
90.0, 101.0,
90.0
90.0, 101.1,
90.0
90.0, 101.4,
90.0
Complexes
per AU
2
2
1
2
2
2
2
2
Resolution
range (Å)a
20–2.25
(2.31–2.25)
20–1.89
(1.93–1.89)
20–2.80
(2.91–2.80)
20–3.20
(3.31–3.20)
20–2.50
(2.57–2.50)
20–2.80
(2.90–2.80)
20–2.70
(2.79–2.70)
20–2.80
(2.89–2.80)
Rmerge (%)
10.7 (75.0)
4.3 (45.0)
6.6 (36.2)
18.3 (39.4)
7.9 (47.6)
8.4 (61.7)
8.7 (73.4)
7.3 (35.3)
I/sI
12.2 (1.7)
25.4 (2.1)
17.9 (3.6)
8.1 (2.0)
18.5 (1.9)
16.8 (2.1)
14.3 (2.6)
15.8 (2.2)
Completeness (%)
98.4 (96.8)
95.9 (79.0)
94.4 (83.0)
96.6 (83.7)
96.7 (75.6)
96.0 (99.9)
98.3 (95.8)
98.6 (91.6)
Redundancy
3.6 (2.8)
3.8 (2.8)
4.6 (3.3)
3.4 (1.9)
4.2 (2.9)
4.1 (3.9)
3.7 (3.6)
4.7 (3.5)
Resolution
range (Å)
20–2.25
20–1.89
20–2.80
20–3.22
20–2.50
20–2.80
20–2.70
20–2.80
Space group
Cell dimensions
Reflections (n)
48,438
81,251
12,442
16,783
35,526
25,336
28,877
25,974
Rfactor/Rfree
19.4/23.5
19.7/23.0
21.7/26.4
22.3/29.4
20.4/23.8
22.6/26.6
20.4/24.7
20.8/25.8
Protein
5480
5480
2740
5480
5480
5480
5480
5480
DNA
1255
1236
627
1181
1089
1096
1073
1096
Ligand (dGTP)
62
62
31
62
62
62
62
62
Ligand (HEPES)
30
—
15
—
15
—
15
—
Atoms (n)
Ion (Ca2+)
6
6
3
6
6
6
6
6
Water
326
616
36
35
129
64
61
64
Protein
26.42
25.05
34.6
47.3
37.8
28.3
33.1
28.3
DNA
30.88
27.17
50.17
78.6
51.5
46.8
37.4
41.0
Ligand (dGTP)
15.6
7.61
34.7
44.2
34.9
27.6
28.5
27.7
Ligand (HEPES)
41.1
—
90.9
—
51.7
—
51.4
—
Ion (Ca2+)
31.5
29.1
46.7
67.26
52.7
47.2
47.6
44.9
Water
32.7
35.8
28.6
26.2
36.5
29.3
27.6
29.0
Bond length (Å)
0.008
0.008
0.009
0.009
0.009
0.009
0.008
0.009
Bond angles ( )
1.42
1.37
1.58
1.44
1.46
1.52
1.44
1.47
B-factors
Rmsd
AU, asymmetric unit; rmsd, root-mean-square deviation. See also Figure S1.
a
Values in parentheses are for highest-resolution shell.
RESULTS
DNA Template/Primer Design for Structure
Determination
To obtain the crystals of the Dpo4 extension ternary complexes
with correct as well as with misinserted 30 terminal primer
bases opposite the m1G and m3C lesions (Figure 1A) we used
50 -CTAAC[X*]C-.30 19-mer templates, where [X*] is m1G or
m3C, and 20 ,30 -dideoxy-Y terminated 13-mer primer strands,
where Y is C, G, T, or A (Figure 1B). An incoming dGTP was
added to pair with the template base C flanking the m1G or
m3C on its 50 -side. The structures of the Dpo4 m1G-modified
(designated m1G,C, m1G,G, m1G,T, m1G,A) and m3Cmodified (m3C,G, m3C,A, m3C,C, m3C,T) ternary complexes
were solved by the molecular replacement method. The crystal
data, together with the data collection and refinement statistics
are summarized in Table 1.
Structure of the m1G,C Extension Ternary Complex
The overall structure and conformation of the m1G,C extension
ternary complex with correct primer C base opposite the lesion
(Figure 1C) is similar (Ca root-mean-square deviation [rmsd] =
0.99 Å) to the type I unmodified ternary complex (Ling et al.,
2001) (see Figure S1 available online). The Dpo4 polymerase
embraces the template/primer DNA by the palm, finger, and
thumb domains, that are present in all known DNA polymerases,
and by the little finger or polymerase associated, PAD, domain
that is unique to the Y-family (Johnson and Beese, 2004; Steitz
Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved 823
Structure
Bypass of Methylation Damage Lesions by Dpo4
m1G•C complex
m1G•T complex
A
C
10.0 Å
2.7 Å
Finger
Tyr12
C5
(0) A4
m1G
(–3)
Little
finger
C7
T8
m1G(anti)
B
dGTP
Asp105
(–1)
(–2)
T14(anti)
Palm
A3
G13
A
Asp7
D
(inside the helix)
m1G•G complex
Glu106
A12
Template
Primer
G14(anti)
C14
m1G(anti)
B
(major groove)
m1G•A complex
E
A14 is disordered
G
m1G(anti)
C
C14
m1G(anti)
(minor groove)
Figure 2. Pairing Alignments of the m1G Lesion with Correct C and Misinserted T, G, or A 30 -Terminal Primer Bases at the (–1) Position of
Extension Ternary Complexes
(A) Structure of the active site of the m1G,C complex. m1G(anti) at the (–1) position is opposite the 30 -terminal C14(anti) base of the primer strand, which is
displaced into the minor groove. The next template base C5 is paired with an incoming dGTP at the (0) position of the active site. Simulated annealing Fo-Fc omit
map contoured at 3s level and colored in blue (2.25 Å resolution) is shown for m1G and C14 residues, and at 2s level, colored in gray, is shown for C14.
(B) m1G opposite C14 of the primer strand (colored sticks). A Watson-Crick pair between an unmodified-G and primer base C (black lines) is shown at the (–1)
position of a Dpo4 complex (PDB ID 2AGQ) (Vaisman et al., 2005); structures are superimposed by Ca atoms of the little finger domains. The CH3 group at the N1
of m1G displaces partner C14. The observed electron densities for the phosphate and sugar groups of C14, and the partial density for its base, is consistent with
positioning of C14 toward the minor grove. Nevertheless, given the poor density for the base, there is uncertainty regarding the precise orientation of the C14 base
and hence is not shown.
(C) m1G(anti) opposite intrahelical T14(anti) of the primer strand of the m1G,T complex. The observed electron density for the phosphate group and partial density
for the base and sugar groups of T14, is consistent with an inside-the-helix location for the T14 residue; however, given the poor density for the sugar and base of
T14, there is uncertainty regarding the precise orientation of this residue, with the intrahelical alignment shown in light coloring.
(D) m1G(anti) opposite G14(anti) of the primer strand of the m1G,G complex.
(E) m1G(anti) opposite disordered A14 of the m1G,A complex. Simulated annealing Fo-Fc omit maps contoured at 3s level and colored in blue are shown for
damaged pairs in the m1G,T, m1G,G, and m1G,A complexes at 1.89 Å, 2.80 Å, and 3.20 Å resolution, respectively; in addition, omit maps at 2s level and colored
in gray are shown for T14 and G14. The overall structural superposition of the m1G,C, m1G,T, m1G,G, m1G,A and unmodified Dpo4 complexes is shown in
Figure S1. See also Figure S2.
and Yin, 2004; Swan et al., 2009a; Yang and Woodgate, 2007).
The polymerase active site ions A and B, identified previously
as Ca2+ under identical crystallization conditions (Rechkoblit
et al., 2006), take analogous positions to the metal ions present
in the ternary complexes of high-fidelity polymerases (Vaisman
et al., 2005). The third ion, coordinated by the loop of the thumb
domain, is found in the majority of the Dpo4 ternary complexes
(Bauer et al., 2007; Eoff et al., 2007; Ling et al., 2001, 2003; Rechkoblit et al., 2006, 2010; Zhang et al., 2009); an ion at a similar
position has been observed in other Y-family polymerases
including human and yeast Rev1 (Swan et al., 2009b), human
Pol k (Lone et al., 2007), and Pol i (Nair et al., 2004).
A nascent base pair at the (0) position of the Dpo4 active site
pocket is formed by the C5 template base (50 adjacent to m1G)
and the incoming dGTP (Figure 2A). The m1G lesion at the (–1)
position of the Dpo4 active site takes an anti conformation and
stacks within the template/primer helix (Figures 2A and 2B).
Such an arrangement of the m1G lesion is similar to that of
a template G base at the same position of an unmodified
Dpo4 complex (Protein Data Bank [PDB] ID 2AGQ) (Vaisman
et al., 2005) (black lines in Figure 2B). We find that clashing
of the methyl group at the N1 of m1G with the partner primer
base C (that would occur as judged by the unmodified complex
[Vaisman et al., 2005]) is avoided due to m1G retaining its
spatial intrahelical Watson-Crick orientation with respect to an
unmodified guanine, but placing the burden of distortion on
the unmodified cytosine-containing opposing strand (black
lines in Figure 2B). Thus, to accommodate the methyl group
in the m1G,C complex, the base, sugar, and phosphate of
C14 are pushed out of the DNA duplex toward the minor
groove, as is evident from the simulated annealing Fo-Fc omit
map shown in Figure 2B. The phosphate and sugar groups of
the partner residue C14 are well-defined in the electron density
map (Figure 2B). However, the density for the base of C14 is
partially disordered (base not shown in Figures 2A and 2B) indicating its conformational flexibility outside the double helix.
Because the sugar of the C14 primer terminus and, thus, its
putative 30 -OH is displaced and not properly positioned for
an extension reaction to occur, the m1G,C complex is catalytically incompetent.
824 Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Bypass of Methylation Damage Lesions by Dpo4
Mutagenic m1G,T, m1G,G, and m1G,A Extension
Complexes
The m1G,T, m1G,G, and m1G,A ternary complexes represent an
extension step from the misinserted T, G, or A primer bases opposite the mutagenic m1G lesion. The overall arrangements of these
complexes and active site alignments (Figures S1 and S2) are
similar to that of the m1G,C nonmutagenic complex (Figures 1C
and 2A). Interestingly, as seen with the m1G,C, all mutagenic
complexes show the m1G lesion to be in the normal anti conformation while maintaining base stacking with the adjacent template
C5 and C7 bases (Figures 2C–2E; Figures S2A–S2C).
In the m1G,T complex, the position of the phosphate group of
the partner primer base T14, that is well-defined in the electron
density map (Figure 2C; Figure S2A), points to an inside-the-helix
location for the T14 residue. However, despite the 1.89 Å resolution, the density for the sugar and base of T14 is poor; this
suggests the possibility of alternate conformation(s) for T14.
For the inside-the-helix location of the m1G,T pair (base and
sugar of T are shown in light color due to the tentative nature
of alignment in Figure 2C), the N2 of m1G could form a single
hydrogen bond with the O2 of T14; this arrangement differs
from the commonly found wobble G,T mismatch, where a T
base is shifted toward the major groove and the N1 and O6 of
G form two hydrogen bonds with the O2 and N3 of T, respectively (Hunter et al., 1987; Patel et al., 1982).
In the m1G,G complex, the partner residue G14 is displaced
into the major groove (Figure 2D) and cannot support catalysis.
Furthermore, in the m1G,A complex, the phosphate, sugar
and base of the partner residue A14 are disordered (Figure 2E).
Similar to G14, A14 is most likely displaced outside the helix,
because modeling of A14 in either anti or syn conformation
with intrahelical occupancy would result in collision with the
m1G base (data not shown).
m3C Extension Complexes Opposite G, A, C, and T
The overall structures and conformations of the m3C-containig
extension ternary complex are similar to each other, to m1Gmodified complexes and to the type I unmodified ternary
complex (Ling et al., 2001) (Figure S3). In contrast to the m1Gmodified extension complexes (Figure 2), the smaller base of
the m3C lesion allows its partner bases G, A, C, and T to enter
the double helix (Figures 3A–3E; Figure S3). However, the electron densities for the base and sugar of the partner G14 and
T14 residues are observed at a lower s level than for the
surrounding entities and, thus, may be indicative of the existence
of alternate conformers (Figures 3B and 3E). For this reason, the
intrahelical positioning of G14 (Figure 2B) and T14 (Figure 4E) are
shown in light color due to the tentative nature of the alignments.
The ‘‘correct’’ m3C,G base pair with an intrahelical orientation
of G is highly sheared, with m3C(anti) shifted into the major
groove and its partner G14(anti) into the minor groove (Figures
3A and 3B). The base stacking interactions between m3C and
the 30 -adjacent C7 template base, as well as between the G14
and G13 primer strand bases are disrupted; however, the base
stacking between the 50 -adjacent C5 template base and the
base of m3C is maintained. The sheared m3C,G pair is held
together by hydrogen bond interactions, with the methyl group
of the positively charged m3C potentially serving as a hydrogen
bond donor to the O6 of G14 (3.0 Å separation; Figure 1B).
In the mutagenic m3C,A, m3C,C, and m3C,T complexes, the
base of the m3C is positioned within the double helix and maintains stacking with the adjacent C5 and C7 template bases
(Figures 3C–3E; Figures S4A–S4C). The C10 -C10 distances
between the m3C and its partner bases are in the range of
9.1 Å to 10.1 Å (Figures 3B–3E), less than the 10.5 Å value for
a normal Watson-Crick pair. In all these complexes, the CH3
group of the m3C lesion could serve as a donor for hydrogen
bonding interactions and is positioned near the acceptor
(Figures 3C–3-E).
Examples of C-H,,,O/N acceptor hydrogen bonds have
been observed in many protein-nucleic acid complexes (Braddock et al., 2002; Mandel-Gutfreund et al., 1998; Wahl and
Sundaralingam, 1997). In these structures, the methyl group of
T or the H5 atom of C serves as donors and oxygen atoms of
amino acids serve as acceptors. In addition, in some structures,
donor-acceptor arrangements have also been found between
the methyl group of isoleucine and the O2 or N3 atoms of C
(Braddock et al., 2002).
Efficiency and Fidelity of Dpo4 Translesion Synthesis
Here we show that Dpo4 is strongly inhibited and predominantly
mutagenic during base insertion opposite the m1G and m3C
lesions. Further extension after insertion of both correct and
incorrect bases is inefficient and introduces additional base
substitution and deletion errors.
Dpo4 readily elongates the 13-mer primer strand using the
unmodified-G or C DNA templates, to produce predominantly
full-length 19-mer extension products in the presence of all
four dNTPs (Figure 4A, lanes 1–5 and 11–15, respectively). In
contrast, primer elongation on the m1G- and m3C-modified
templates is inefficient and produces only up to 14% of fully
extended products after 20 min reaction time with a higher
concentration of Dpo4 (Figure 4A, lanes 6–10 and 16–20, respectively). Single base incorporation experiments (Figure 4B) indicate that Dpo4 generates 90% mutations during the insertion
step opposite m1G and 60% opposite m3C, when calculated
as the ratio of correctly inserted nucleotide over all inserted
nucleotides. Surprisingly, the efficiencies of correct as well as
incorrect base insertions opposite m1G and m3C lesions are
smaller than the efficiencies of misincorporation opposite
unmodified G or C. This is evident from a comparison of A, G,
and T misincorporation opposite template G for 5 min under
10 nM Dpo4 with base insertion opposite m1G under the same
conditions (Figure S5B); the same results are observed for misincorporation opposite template C and base insertion opposite
m3C (Figure S5B). Therefore, we conclude that the efficiencies
of C, A, G, and T incorporation opposite m1G and m3C are
reduced by more than 104- to 105-fold as compared to incorporation of the correct dNTPs opposite unmodified template
bases (Rechkoblit et al., 2006; Zhang et al., 2009). We were
not able to evaluate steady-state kinetic parameters for base
insertions opposite the m1G and m3C lesions due to slow reaction rates that required high Dpo4 concentrations to reliably
quantify the products; a high enzyme to substrate ratio is incompatible with the Michaelis-Menten kinetics (Creighton et al.,
1995).
Primer extension from the correctly paired unmodified G,C
and C,G termini is robust; only single bands corresponding to
Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved 825
Structure
Bypass of Methylation Damage Lesions by Dpo4
m3C•A complex
m3C•G complex
C
9.5 Å
A
Finger
2.7 Å
Tyr12
m3C(anti)
A14(anti)
Palm
A3
C5
A4
dGTP
Little
finger
G13
C7
m3C•C complex
B
m3C G14 Asp105 A
Asp7
D
9.1 Å
Glu106
A12
T8
3.0 Å
Template
Primer
m3C(anti)
B
C14(anti)
10.0 Å
m3C•T complex
E
10.1 Å
2.9 Å
3.0 Å 2.9 Å
3.4 Å
m3C(anti)
3.5 Å
G14(anti)
m3C(anti)
3.0 Å
2.8 Å
T14(anti)
Figure 3. Pairing Alignments of the m3C Lesion with Correct G and Misinserted A, C, or T 30 -Terminal Primer Bases at the (–1) Position of
Extension Ternary Complexes
(A) Structure of the active site of the m3C,G complex. m3C(anti) at the (–1) position is opposite the 30 -terminal G14(anti) base of the primer strand. Simulated
annealing Fo-Fc omit map contoured at 3s level and colored in blue (2.80 Å resolution) is shown for m3C and G14 residues, and at 2s level colored in gray, is
shown for G14.
(B) m3C opposite correct G14 of the primer strand. The electron density for the base of G14 is not well defined, thus the base of G14 is shown in light coloring due
to an uncertainty regarding its precise orientation.
(C) m3C(anti) opposite A14(anti) of the primer strand of the m3C,A complex.
(D) m3C(anti) opposite C14(anti) of the primer strand of the m3C,C complex.
(E) m3C(anti) opposite T14(anti) of the m3C,T complex. Given the poor density for the sugar and base of T14, there is uncertainty regarding the precise orientation
of this residue, with the intrahelical alignment shown in light coloring. Simulated annealing Fo-Fc omit maps contoured at 3s level and colored in blue are shown
for damaged pairs in the m3C,A, m3C,C, and m3C,T complexes at 2.70 Å, 2.50 Å, and 2.80 Å resolution, respectively; in addition, omit map at 2s level and
colored in gray is shown for T14. Water molecules are shown as small red spheres. The overall structural superposition of the m3C,G, m3C,A, m3C,C, m3C,T
and unmodified Dpo4 complexes is shown in Figure S3. See also Figure S4.
partially extended 15-, 16-, 17- and 18-mers are evident (Figure 4C, lanes 1–2 and 21–22; Figure S6, top). In contrast, with
correct m1G,C and m3C,G termini (Figure 4C, lanes 9–10 and
29–30), as well as with mismatched damaged termini (Figure 4C,
lanes 11–14 and 25–28, 31–32), extension efficiency is low;
furthermore, it is less than that from the mismatched A and G
opposite the template G (Figure 4C, lanes 3–6) and from the mismatched C, A, and T opposite the template C (Figure 4C, lanes
17–20 and 23–24). These results indicate that the extension
from m1G,C and m3C,G, as well as from the damaged
mismatches, is at least 103-fold less efficient than from the
unmodified matched termini (Rechkoblit et al., 2006; Trincao
et al., 2004). Interestingly, there was a significant amount of
Dpo4 primer extension from the mismatched m1G,T pair, with
extension rates from m1G,T (Figure 4C, lanes 15–16) and G,T
(Figure 4C, lanes 7–8) mismatches appearing to be comparable.
Moreover, during primer elongation from correctly matched
m1G,C and m3C,G, as well as ‘‘incorrectly’’ paired m3C,C
termini, additional bands for 15-, 16-, 17-, and 18-mers are de-
tected with respect to the unmodified G or C template, indicating
mutagenic extension (Figure 4C: lanes 9–10 versus 1–2, lanes
29–30 versus 21–22, and lanes 25–26 versus 17–18; Figure S6,
bottom). This reflects substitution errors that arose during extension from the correctly paired and mismatched damaged termini
and, thus, indicates that m1G and m3C lesions can also cause
semi-targeted substitution mutations. In addition, a predominant
18-mer product is detected during extension from m3C,C
termini (Figure 4C, lanes 25–26) suggesting a semi-targeted
deletion mutation.
DISCUSSION
Dpo4-Catalyzed Insertion Opposite m1G and m3C
The structures of the Dpo4 complexes reveal that during extension, both the m1G and the m3C lesions stay within the DNA
template/primer helix and present their Watson-Crick edges
(sterically compromised by the bulky methyl group) for base pairing. This causes either eviction of m1G’s correct partner C from
826 Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Bypass of Methylation Damage Lesions by Dpo4
the double helix into the minor groove (Figure 2B), or displacement of m3C’s correct partner G into the minor groove (Figure 3B); the incorrect partner bases are also primarily evicted
opposite m1G or misaligned opposite m3C. During the insertion
step, these positions of m1G and m3C would hinder entrance of
a dNTP into the Dpo4 active site, consistent with pronounced
primer terminal nucleotide distortion (Figures 2 and 3). This is
consistent with the reduced insertion (by more than 104-fold
to 105-fold) of both correct and incorrect dNTPs opposite m1G
and m3C compared to incorporation of correct nucleotides
opposite unmodified template bases.
Dpo4’s Catalytic Feasibility for Extension Past m1G
and m3C Lesions
In the m1G-modified Dpo4 complexes, further extension from
the primer bases C, G, and A in their extrahelical positions
(Figures 2A, 2B, 2D, and 2E) would not be possible. However,
extension from m1G,C, m1G,G, and m1G,A termini still occurs,
although with very low efficiency, and results in correct, as well
as mutagenic extension products (Figure 4C). To explain this
we hypothesize that for correct extension these primer residues
would enter the helix and position their 30 -OH groups for elongation, and thereby dislocate the m1G from its internal helical position (these rare events are not captured in the crystal structures).
The observed mutagenic extension could arise due to pairing of
the template base 50 to the m1G with the 30 -terminal primer base,
so as to skip m1G; a 1 frameshift complex, similar to the one
observed with an abasic lesion (Ling et al., 2004), could potentially form. Alternatively, semi-targeted misalignment events
are possible as proposed earlier for the bulky 2-aminofluorenedG lesion (Rechkoblit et al., 2010).
Interestingly, extension from a T base opposite m1G occurs
faster than from the m1G,C pair (Figure 4C). The observed intrahelical position of the partner T base (at lower s level) appears to
be feasible for extension (Figure 2C). Even though the 30 -carbon
of the sugar of the primer terminal T14 in m1G,T (the 30 -OH is
absent) is relocated by 1.6 Å toward the major groove
compared to the 30 -C position of C in the unmodified G,C
complex (Figure S7), the distance between the 30 -carbon of
T14 and the a-phosphate of dGTP is 3.8 Å, similar to the analogous distance in the unmodified complex with a 20 , 30 -dideoxy
primer terminus (4.2 Å, PDB ID 2AGQ) (Vaisman et al., 2005).
However, the catalytic cation A, which coordinates the 30 -OH
group and activates it for reaction (Steitz and Yin, 2004) is repositioned by 2.2 Å relative to its location in the unmodified
complex (Figure S7). The A-site metal ion has often been found
to be displaced in Dpo4 complexes that contain inhibiting
mismatches or lesions (Rechkoblit et al., 2010; Vaisman et al.,
2005; Yang and Woodgate, 2007). It has been suggested that
the correct geometry of ion binding is critical for the chemical
bond formation step and only a suitable substrate allows the
ion to be properly positioned (Vaisman et al., 2005; Yang and
Woodgate, 2007). Thus, repositioning of the A ion is consistent
with inhibition of extension from T14 with respect to an unmodified complex.
In the m3C-modified complexes the 30 -primer termini of the G,
A, C, and T partner bases relocate by no more than 1.0 Å relative to the position observed in the unmodified G,C complex
(Figures 5A and 5B). Hence, the distances from the 30 -carbon
of the sugar to the a-phosphate of dGTP are in a productive
3.8 to 4.2 Å range. However, for all four complexes, the catalytic
cation A repositions by 2.5 Å relative to its location in the
unmodified complex (Figure 5). Thus, efficient catalysis is not
promoted, allowing time for misalignment events to occur.
m3C and G Form a Pair with CH3 of m3C Acting
as a Hydrogen Bond Donor
Our observed m3C,G pair with a 10.0 Å C10 -C10 distance (Figure 3B) features a novel hydrogen bond where the methyl group
of m3C serves as a donor. Consistently, the CH3 group forms
hydrogen bonds with acceptor atoms of A, C, and possibly T
bases (Figures 3C–3E). By contrast, a different positioning of
m3C opposite G was reported for the m3C,G alignment in
duplex DNA using a protein-linked protein-DNA system (Lu
et al., 2010). In this structure, both m3C and opposing G are
shifted toward the major and minor grooves respectively, but
are not hydrogen-bonded to each other, and exhibit a long
14.5 Å C10 -C10 separation. This distinctly different alignment
most likely reflects contributions resulting from the disruption
of two base pairs that are immediately adjacent to the m3C,G
in this structure; the T base 30 to the m3C skips its partner A
and forms a noncanonical pair with the next A, whereas the C,
which follows the T, is crosslinked to the protein and flips out
of the double helix.
Proposed Model for ABH2 Recognition of Alkylated
DNA Lesions
The lesion search mechanism employed by the repair demethylase ABH2 is unclear and is of great interest in the field. To elucidate this process, structures of cognate m1A,T and m3C,G
alignments within DNA duplexes have been investigated (Lu
et al., 2010) and m1A has been shown to adopt a syn conformation presenting its Hoogsteen edge to form a base pair with T; the
latter shifted from its position in a normal A(anti),T Watson-Crick
pair (C10 -C10 distance of 10.5 Å) to compensate for the shorter
9 Å distance between C10 of m1A(syn) and C10 of T.
We find that a common theme in our observed correct
m1G(anti),C and m3C(anti),G pairs, as well as the m1A(syn),T
pair (Lu et al., 2010), is the displacement of the partner C, G
and T bases from their respective normal positions in duplex
DNA. Thus, it is tempting to propose that ABH2 employs interactions with the complementary undamaged strand of the DNA
duplex while scanning DNA for lesions (ABH2 is known to prefer
dsDNA substrates (Aas et al., 2003; Duncan et al., 2002; Falnes,
2004; Ringvoll et al., 2006)). Such a lesion recognition strategy
has been proposed for the glycosylase AlkD (Rubinson et al.,
2010) and the nucleotide excision repair protein Rad4/XPC
(Min and Pavletich, 2007).
Incorrect Partner Bases Opposite m3C Could
Complicate Recognition by ABH2
Generally, DNA glycosylases, that flip alkylated bases, exhibit
enhanced excision activity for lesions in distorted mispaired
arrangements (O’Brien and Ellenberger, 2004). We observe
that the positions of the bases in the correct m3C,G pair deviate
the most from ones in the unmodified C,G pair compared to the
mutagenic m3C,A, m3C,C, and m3C,T alignments (Figure 5B).
This leads us to speculate that ABH2 could detect m3C in the
Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved 827
Structure
Bypass of Methylation Damage Lesions by Dpo4
Elongation from a primer base positioned before the modification site
1 2 3 4 5
6
7 8 9 10 11 12 13 14 15 16 17 18 19
5 - C T A A C [X*] C T A C C A T C C A A C C -3
G A T G G T A G G T T G G -5
19 18 17 16 15 14
[X*] = G, m1G, C, or m3C
13 12 11 10 9 8 7 6 5 4 3 2 1
Primer elongation reaction: all dNTPs
A
Unmodified G
1
3
m1G
Unmodified C
1 3 5 10 20
5 10 20
1 3
m3C
5 10 20
1
3
5 10 20 min
19-mer-
-19-mer
18-mer-
-18-mer
17-mer-
-17-mer
16-mer-
-16-mer
-15-mer
15-mer-
-14-mer
14-mer-
-13-mer
13-merLane no. 1
2
3
4
5
6 7 8 9 10
11 12 13 14 15
16 17 18 19 20
Single base incorporation opposite:
B
Unmodified G
5 min
C A G
Reaction time:
dNTP:
T
m1G
Unmodified C
m3C
20 min
C A G T
5 min
C A G T
20 min
C A G T
-15-mer
-14-mer
14-mer-
-13-mer
13-mer-
Extension from a primer base opposite the modification site
C
1 2 3 4 5
6
7
5 - C T A A C [X*] C... -3
Y G... -5
19 18 17 16 15
14 13
Unmodified G
Template:
template
primer
all dNTPs
m1G
A
G
T
Primer:
C
Reaction
5 20 5 20 5 20 5 20
time:
C
A
[X*] = G, m1G, C, or m3C
Y = C, A, G or T
Unmodified C
G
T
5 20 5 20 5 20 5 20
C
A
5 20 5 20
m3C
G
T
5 20
5 20
C
A
5 20 5 20
G
T
5 20 5 20 min
-+1 addition
+1 addition-
-19-mer
19-mer-18-mer
18-mer-
-17-mer
17-mer-
-16-mer
16-mer-
-15-mer
15-mer-14-mer
14-merLane no. 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
17 18 19 20 21 22 23 24
25 26 27 28 29 30 31 32
Figure 4. Efficiency and Fidelity of Base Incorporation and Extension of Primers Bound to Unmodified-G, m1G-, Unmodified-C, and
m3C-Templates by Dpo4
(A) Time course of extension of 32P 50 -end-labeled 13-mer primers bound to 19-mer templates in the presence of all four dNTPs. The 30 -end of the 13-mer primer
was paired with the base on the 30 -side of the unmodified-G, m1G-, unmodified-C, or m3C template. The reactions were conducted with 10 nM Dpo4 on the
828 Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Bypass of Methylation Damage Lesions by Dpo4
A
B
m3C
G
Asp105
B
Tyr12
Asp105
Glu106
Asp7
A
Asp7
C
G
A
G14
C
Glu106
G14
m3C
m3C•G
m3C•A
m3C•C
m3C•T
unmodified G•C
Figure 5. Superposition of the m3C,G, m3C,A, m3C,C, m3C,T and Unmodified Dpo4 Complexes
(A) View of 50 -.(C5-m3C-C7) template and 30 -Y14-G13 (Y = G, A, C, or T) primer segments plus dGTP and equivalent entities of the unmodified complex (PDB ID
2AGQ) (Vaisman et al., 2005). See labels for color coding. Structures are superimposed by Ca atoms of the active site forming palm and finger domains of Dpo4.
Asp7, Asp105, Glu106, and Tyr12 are shown in sticks for the m3C,G (pink) and unmodified (silver) complexes. In all four m3C-modified complexes the catalytic
cation A, shown as a color-coded sphere, repositions by 2.5 Å relative to its location in the unmodified complex (silver sphere).
(B) View of m3C-containing and unmodified base pairs looking down the helix axis.
correct m3C,G pair more efficiently than in the incorrect base
pairs resulting in longer persistence of this lesion opposite
mismatches. If true, this would suggest a mechanism whereby
a mutagenic insertion opposite m3C may have another chance
of being removed by the exonuclease domain of a high-fidelity
polymerase (or perhaps by another DNA repair protein) before
making such a mutation permanent by repair of m3C,(A, C, or
T) to C,(A, C, or T) mismatches by ABH2, thus increasing the
overall fidelity of replication. This would be especially important
if mismatch repair was lost, as seen in various tumor cells
(Karamurzin and Rutgers, 2009; Poulogiannis et al., 2010). This
paradigm would be analogous to the GO system by which the
E. coli DNA glycosylase MutM and its human homolog OGG1
will only excise 8-oxoguanine if the opposite base is cytosine,
whereas another repair glycosylase (E. coli MutY and human
MYH) will remove mismatched adenine opposite 8-oxoguanine
(Fromme et al., 2004).
most likely to be inefficient during insertion opposite m1G and
m3C. In contrast, Pol i that rotates adenine (Nair et al., 2004)
and guanine (Nair et al., 2005) templating bases into the syn
conformation to employ their Hoogsteen edges for dNTP
binding, might be able to bypass the m1G lesion. However,
structural (Jain et al., 2009) and kinetic (Choi et al., 2009) data
suggest that Pol i engages the Watson-Crick edge of template
pyrimidines for base pairing with dNTP, thus presenting a challenge for base insertion opposite the m3C. In addition, Rev1
polymerase, which displays template G outside the DNA helix
and uses an arginine residue to pair with incoming dCTP
(Swan et al., 2009b), might be efficient in bypass of m1G. Future
experimental investigations, both structural and biochemical,
are needed to further elucidate the functions of the human polymerases in processing these lesions.
Bypass of m1G and m3C by Human Y-Family
Polymerases
Our results provide opportunities for proposing hypotheses concerning processing of these alkylated lesions by human bypass
polymerases. We propose that Pol k (Lone et al., 2007) and Pol h
(Biertumpfel et al., 2010), which, like Dpo4, employ WatsonCrick base pairing between the templating base and dNTP, are
m1G- and m3C-Modified DNA Templates
m1G- and m3C-modified 19-mer oligonucleotides (Figure 1B) were synthesized by automated solid-phase synthesis, deprotected with concentrated
ammonium hydroxide, purified by anion exchange high-performance liquid
chromatography (HPLC), and characterized by matrix-assisted laser desorption/ionization essentially as described previously (Delaney and Essigmann,
2004); the m1G and m3C oligonucleotides were deprotected at 37 C for 4 hr
(m1G) or 12 hr (m3C) before lyophilization and HPLC purification.
EXPERIMENTAL PROCEDURES
unmodified G- and C-templates and with 50 nM Dpo4 on the m1G- and m3C-templates; the concentration of template/primer DNAs was 10 nM. See Figure S5A
for the experiments conducted on the damaged templates with 10 nM Dpo4.
(B) dCTP, dATP, dGTP, or dTTP single nucleotide insertion. The green triangles represent insertion of the correct nucleotide; the magenta triangles represent
insertion of an incorrect nucleotide. The reactions were conducted for 5 min with 10 nM Dpo4 on the unmodified G- and C-templates and for 20 min with 50 nM
Dpo4 on the m1G- and m3C-templates. See Figure S5B for the experiments conducted for 5 min with 10 nM Dpo4 on the damaged templates. The percentages of
C, A, G, and T incorporation into the primer strand on the unmodified-G template were 93%, 6.3%, 1.9%, and 4.9% respectively, and on the unmodified-C
template were 2.5%, 5.5%, 95%, and 5.8%, respectively. Note that two G bases are correctly incorporated opposite template C and 50 -adjacent C template
bases. The percentages of C, A, G, and T incorporation into the primer strand on the m1G template were 2.2%, 5.6%, 9.8%, and 2.5%, respectively, and on the
m3C template were 5.2%, 11%, 12%, and 1.8%, respectively.
(C) Efficiency of extension from C, A, G, and T bases opposite the unmodified-G, m1G, unmodified-C and m3C by Dpo4 (see labels). Additional 15-, 16-, 17-, and
18-mer bands that migrate with different mobilities than the correctly elongated bands arising from the extension from matched unmodified G,C and C,G termini,
are detected, thus indicating mutagenic extension. An overexposed image of the gel is used to show mutagenic primer extension events. The green triangles
represent the correctly extended products; the magenta triangles represent mutagenic extension. 20-mer products indicate (+1) nontemplate directed addition
that is commonly observed with different DNA polymerases. Profile analysis of 32P-signal intensities in lanes 2, 10, 22, and 30 of the gel image exposed within
the linear dynamic range of the PhosphorImager system is shown in Figure S6. See also Figure S5.
Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved 829
Structure
Bypass of Methylation Damage Lesions by Dpo4
Crystallization
The crystals of the Dpo4 extension ternary complexes containing m1Gor m3C-modified 19-mer templates and 13-mer primers terminated with
20 ,30 -dideoxy-C, 20 ,30 -dideoxy-A, 20 ,30 -dideoxy-G, or 20 ,30 -dideoxy-T (Figure 1B) opposite m1G- or m3C- lesions were grown in the presence of
dGTP and flash-frozen in liquid nitrogen for X-ray data collection under conditions described previously (Rechkoblit et al., 2006). Several rounds of micro
seeding were employed to produce the diffraction quality crystals.
We were not able to obtain crystals of the insertion ternary complexes with
dCTP, dATP, dGTP, or dTTP opposite the m1G or m3C and a primer terminated with 20 ,30 -dideoxy-G opposite a C7 base, 30 to the lesion site.
Structure Determination and Refinement
X-ray diffraction data were collected at the NE-CAT 24-ID-C beam line at the
Advanced Photon Source (Argonne National Laboratory, Chicago). The data
were processed and scaled using the HKL2000 suite. The structures of the
extension complexes were solved by the molecular replacement method,
using our published oxoG-modified extension ternary Dpo4-DNA-dGTP structure (Rechkoblit et al., 2009) as a search model. The model building was manually finished in TURBO-FRODO (http://www.afmb.univ-mrs.fr/-TURBO-)
based on the electron density maps calculated in REFMAC (http://www.
ccp4.ac.uk/html/refmac5.html), and the resulting models were refined in REFMAC. The crystal data, together with the data collection and refinement
statistics for all structures are summarized in Table 1. The simulated annealing
omit maps were calculated in CNS (http://cns-online.org/v1.21/) with the m1G,
m3C and partner bases omitted from the models before they were heated to
2000 K and then slowly cooled.
Primer Elongation Assays
Primer elongation assays were conducted as described by our group in the
literature (Rechkoblit et al., 2006).
ACCESSION NUMBERS
REFERENCES
Aas, P.A., Otterlei, M., Falnes, P.O., Vagbo, C.B., Skorpen, F., Akbari, M.,
Sundheim, O., Bjoras, M., Slupphaug, G., Seeberg, E., and Krokan, H.E.
(2003). Human and bacterial oxidative demethylases repair alkylation damage
in both RNA and DNA. Nature 421, 859–863.
Allan, J.M., and Travis, L.B. (2005). Mechanisms of therapy-related carcinogenesis. Nat. Rev. Cancer 5, 943–955.
Aravind, L., and Koonin, E.V. (2001). The DNA-repair protein AlkB, EGL-9, and
leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biol. 2, research0007.1–research0007.8.
Bauer, J., Xing, G., Yagi, H., Sayer, J.M., Jerina, D.M., and Ling, H. (2007). A
structural gap in Dpo4 supports mutagenic bypass of a major benzo[a]pyrene
dG adduct in DNA through template misalignment. Proc. Natl. Acad. Sci. USA
104, 14905–14910.
Biertumpfel, C., Zhao, Y., Kondo, Y., Ramon-Maiques, S., Gregory, M., Lee, J.Y.,
Masutani, C., Lehmann, A.R., Hanaoka, F., and Yang, W. (2010). Structure and
mechanism of human DNA polymerase eta. Nature 465, 1044–1048.
Boiteux, S., and Laval, J. (1982). Mutagenesis by alkylating agents: coding
properties for DNA polymerase of poly (dC) template containing 3-methylcytosine. Biochimie 64, 637–641.
Braddock, D.T., Baber, J.L., Levens, D., and Clore, G.M. (2002). Molecular
basis of sequence-specific single-stranded DNA recognition by KH domains:
solution structure of a complex between hnRNP K KH3 and single-stranded
DNA. EMBO J. 21, 3476–3485.
Broyde, S., Wang, L., Rechkoblit, O., Geacintov, N.E., and Patel, D.J. (2008).
Lesion processing: high-fidelity versus lesion-bypass DNA polymerases.
Trends Biochem. Sci. 33, 209–219.
Choi, J.Y., Lim, S., Eoff, R.L., and Guengerich, F.P. (2009). Kinetic analysis of
base-pairing preference for nucleotide incorporation opposite template pyrimidines by human DNA polymerase iota. J. Mol. Biol. 389, 264–274.
Creighton, S., Bloom, L.B., and Goodman, M.F. (1995). Gel fidelity assay
measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion
bypass efficiencies. Methods Enzymol. 262, 232–256.
Coordinates and structure-factor amplitudes for the m1G,C, m1G,T, m1G,G,
m1G,A, m3C,G, m3C,A, m3C,C, and m3C,T complexes have been deposited in the Protein Data Bank with accession codes 3RAQ, 3RAX, 3RB0, 3RB3,
3RB4, 3RB6, 3RBD, and 3RBE, respectively.
De Bont, R., and van Larebeke, N. (2004). Endogenous DNA damage in humans: a review of quantitative data. Mutagenesis 19, 169–185.
SUPPLEMENTAL INFORMATION
Duncan, T., Trewick, S.C., Koivisto, P., Bates, P.A., Lindahl, T., and Sedgwick,
B. (2002). Reversal of DNA alkylation damage by two human dioxygenases.
Proc. Natl. Acad. Sci. USA 99, 16660–16665.
Supplemental Information includes seven figures and can be found with this
article online at doi:10.1016/j.str.2011.03.020.
ACKNOWLEDGMENTS
Delaney, J.C., and Essigmann, J.M. (2004). Mutagenesis, genotoxicity, and
repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli. Proc. Natl. Acad. Sci. USA 101, 14051–14056.
Eoff, R.L., Irimia, A., Egli, M., and Guengerich, F.P. (2007). Sulfolobus solfataricus DNA polymerase Dpo4 is partially inhibited by ‘‘wobble’’ pairing between
O6-methylguanine and cytosine, but accurate bypass is preferred. J. Biol.
Chem. 282, 1456–1467.
We thank Dr. Yuan Cheng in the Patel laboratory for help with expression and
purification of Dpo4 and Dr. Suse Broyde of New York University for extensive
discussions. The research was supported by National Institutes of Health
grants CA46533 to D.J.P., and CA80024 to J.M.E. We thank the staff at the
Northeastern Collaborative Access Team beamlines of the Advanced Photon
Source (APS), Argonne National Laboratory, supported by award RR-15301
from the National Center for Research Resources at the National Institutes
of Health, for assistance with data collection. Use of the Advanced Photon
Source is supported by the U.S. Department of Energy, Office of Basic Energy
Sciences (DE-AC02-06CH11357). O.R. conducted and interpreted all the
structural and primer extension assay data under the supervision of D.J.P;
J.C.D. was responsible for the synthesis and purification of m1G- and m3Ccontaining DNAs under the supervision of J.M.E. The study was written by
O.R. with contributions by D.J.P., J.C.D., and J.M.E.
Falnes, P.O. (2004). Repair of 3-methylthymine and 1-methylguanine lesions
by bacterial and human AlkB proteins. Nucleic Acids Res. 32, 6260–6267.
Received: January 24, 2011
Revised: March 16, 2011
Accepted: March 22, 2011
Published: June 7, 2011
Fu, Y., Dai, Q., Zhang, W., Ren, J., Pan, T., and He, C. (2010b). The AlkB
domain of mammalian ABH8 catalyzes hydroxylation of 5-methoxycarbonylmethyluridine at the wobble position of tRNA. Angew. Chem. Int. Ed. Engl.
49, 8885–8888.
Falnes, P.O., Johansen, R.F., and Seeberg, E. (2002). AlkB-mediated oxidative
demethylation reverses DNA damage in Escherichia coli. Nature 419, 178–182.
Fischer, J., Koch, L., Emmerling, C., Vierkotten, J., Peters, T., Bruning, J.C.,
and Ruther, U. (2009). Inactivation of the Fto gene protects from obesity.
Nature 458, 894–898.
Fromme, J.C., Banerjee, A., Huang, S.J., and Verdine, G.L. (2004). Structural
basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine
DNA glycosylase. Nature 427, 652–656.
Fu, D., Brophy, J.A., Chan, C.T., Atmore, K.A., Begley, U., Paules, R.S., Dedon,
P.C., Begley, T.J., and Samson, L.D. (2010a). Human AlkB homolog ABH8 Is
a tRNA methyltransferase required for wobble uridine modification and DNA
damage survival. Mol. Cell. Biol. 30, 2449–2459.
830 Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Bypass of Methylation Damage Lesions by Dpo4
Gerken, T., Girard, C.A., Tung, Y.C., Webby, C.J., Saudek, V., Hewitson, K.S.,
Yeo, G.S., McDonough, M.A., Cunliffe, S., McNeill, L.A., et al. (2007). The
obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic
acid demethylase. Science 318, 1469–1472.
Han, Z., Niu, T., Chang, J., Lei, X., Zhao, M., Wang, Q., Cheng, W., Wang, J.,
Feng, Y., and Chai, J. (2010). Crystal structure of the FTO protein reveals basis
for its substrate specificity. Nature 464, 1205–1209.
Hecht, S.S. (1999). DNA adduct formation from tobacco-specific N-nitrosamines. Mutat. Res. 424, 127–142.
Huff, A.C., and Topal, M.D. (1987). DNA damage at thymine N-3 abolishes
base-pairing capacity during DNA synthesis. J. Biol. Chem. 262, 12843–
12850.
Hunter, W.N., Brown, T., Kneale, G., Anand, N.N., Rabinovich, D., and
Kennard, O. (1987). The structure of guanosine-thymidine mismatches in BDNA at 2.5-A resolution. J. Biol. Chem. 262, 9962–9970.
Jain, R., Nair, D.T., Johnson, R.E., Prakash, L., Prakash, S., and Aggarwal, A.K.
(2009). Replication across template T/U by human DNA polymerase-iota.
Structure 17, 974–980.
Jia, G., Yang, C.G., Yang, S., Jian, X., Yi, C., Zhou, Z., and He, C. (2008).
Oxidative demethylation of 3-methylthymine and 3-methyluracil in singlestranded DNA and RNA by mouse and human FTO. FEBS Lett. 582, 3313–
3319.
Johnson, S.J., and Beese, L.S. (2004). Structures of mismatch replication
errors observed in a DNA polymerase. Cell 116, 803–816.
Karamurzin, Y., and Rutgers, J.K. (2009). DNA mismatch repair deficiency in
endometrial carcinoma. Int. J. Gynecol. Pathol. 28, 239–255.
Larson, K., Sahm, J., Shenkar, R., and Strauss, B. (1985). Methylation-induced
blocks to in vitro DNA replication. Mutat. Res. 150, 77–84.
Ling, H., Boudsocq, F., Woodgate, R., and Yang, W. (2001). Crystal structure
of a Y-family DNA polymerase in action: a mechanism for error-prone and
lesion-bypass replication. Cell 107, 91–102.
Ling, H., Boudsocq, F., Plosky, B.S., Woodgate, R., and Yang, W. (2003).
Replication of a cis-syn thymine dimer at atomic resolution. Nature 424,
1083–1087.
Ling, H., Boudsocq, F., Woodgate, R., and Yang, W. (2004). Snapshots of
replication through an abasic lesion; structural basis for base substitutions
and frameshifts. Mol. Cell 13, 751–762.
Lone, S., Townson, S.A., Uljon, S.N., Johnson, R.E., Brahma, A., Nair, D.T.,
Prakash, S., Prakash, L., and Aggarwal, A.K. (2007). Human DNA polymerase
kappa encircles DNA: implications for mismatch extension and lesion bypass.
Mol. Cell 25, 601–614.
Lu, L., Yi, C., Jian, X., Zheng, G., and He, C. (2010). Structure determination of
DNA methylation lesions N1-meA and N3-meC in duplex DNA using a crosslinked protein-DNA system. Nucleic Acids Res. 38, 4415–4425.
Mandel-Gutfreund, Y., Margalit, H., Jernigan, R.L., and Zhurkin, V.B. (1998). A
role for CH.O interactions in protein-DNA recognition. J. Mol. Biol. 277,
1129–1140.
McCulloch, S.D., and Kunkel, T.A. (2008). The fidelity of DNA synthesis by
eukaryotic replicative and translesion synthesis polymerases. Cell Res. 18,
148–161.
Park, D.M., Sathornsumetee, S., and Rich, J.N. (2010). Medical oncology:
treatment and management of malignant gliomas. Nat. Rev. Clin. Oncol 7,
75–77.
Patel, D.J., Kozlowski, S.A., Marky, L.A., Rice, J.A., Broka, C., Dallas, J.,
Itakura, K., and Breslauer, K.J. (1982). Structure, dynamics, and energetics
of deoxyguanosine. thymidine wobble base pair formation in the self-complementary d(CGTGAATTCGCG) duplex in solution. Biochemistry 21, 437–444.
Poulogiannis, G., Frayling, I.M., and Arends, M.J. (2010). DNA mismatch repair
deficiency in sporadic colorectal cancer and Lynch syndrome. Histopathology
56, 167–179.
Rechkoblit, O., Malinina, L., Cheng, Y., Kuryavyi, V., Broyde, S., Geacintov,
N.E., and Patel, D.J. (2006). Stepwise translocation of Dpo4 polymerase during
error-free bypass of an oxoG lesion. PLoS Biol. 4, e11.
Rechkoblit, O., Malinina, L., Cheng, Y., Geacintov, N.E., Broyde, S., and Patel,
D.J. (2009). Impact of conformational heterogeneity of OxoG lesions and
their pairing partners on bypass fidelity by Y family polymerases. Structure
17, 725–736.
Rechkoblit, O., Kolbanovskiy, A., Malinina, L., Geacintov, N.E., Broyde, S., and
Patel, D.J. (2010). Mechanism of error-free and semitargeted mutagenic
bypass of an aromatic amine lesion by Y-family polymerase Dpo4. Nat.
Struct. Mol. Biol. 17, 379–388.
Ringvoll, J., Nordstrand, L.M., Vagbo, C.B., Talstad, V., Reite, K., Aas, P.A.,
Lauritzen, K.H., Liabakk, N.B., Bjork, A., Doughty, R.W., et al. (2006). Repair
deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA. EMBO J. 25, 2189–2198.
Rubinson, E.H., Gowda, A.S., Spratt, T.E., Gold, B., and Eichman, B.F. (2010).
An unprecedented nucleic acid capture mechanism for excision of DNA
damage. Nature 468, 406–411.
Saffhill, R. (1984). Differences in the promutagenic nature of 3-methylcytosine as
revealed by DNA and RNA polymerising enzymes. Carcinogenesis 5, 691–693.
Sedgwick, B. (1997). Nitrosated peptides and polyamines as endogenous
mutagens in O6-alkylguanine-DNA alkyltransferase deficient cells.
Carcinogenesis 18, 1561–1567.
Sedgwick, B. (2004). Repairing DNA-methylation damage. Nat. Rev. Mol. Cell
Biol. 5, 148–157.
Sedgwick, B., Bates, P.A., Paik, J., Jacobs, S.C., and Lindahl, T. (2007). Repair
of alkylated DNA: recent advances. DNA Repair (Amst.) 6, 429–442.
Shrivastav, N., Li, D., and Essigmann, J.M. (2010). Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation. Carcinogenesis
31, 59–70.
Silverstein, T.D., Johnson, R.E., Jain, R., Prakash, L., Prakash, S., and
Aggarwal, A.K. (2010). Structural basis for the suppression of skin cancers
by DNA polymerase eta. Nature 465, 1039–1043.
Singer, B., and Grunberger, D. (1983). Molecular Biology of Mutagens and
Carcinogens: Intrinsic Properties of Nucleic Acids (New York: Plenum
Press), 15–44.
Steitz, T.A., and Yin, Y.W. (2004). Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases. Philos. Trans. R. Soc. Lond. B
Biol. Sci. 359, 17–23.
Min, J.H., and Pavletich, N.P. (2007). Recognition of DNA damage by the Rad4
nucleotide excision repair protein. Nature 449, 570–575.
Sundheim, O., Vagbo, C.B., Bjoras, M., Sousa, M.M., Talstad, V., Aas, P.A.,
Drablos, F., Krokan, H.E., Tainer, J.A., and Slupphaug, G. (2006). Human
ABH3 structure and key residues for oxidative demethylation to reverse
DNA/RNA damage. EMBO J. 25, 3389–3397.
Nair, D.T., Johnson, R.E., Prakash, L., Prakash, S., and Aggarwal, A.K. (2005).
Human DNA polymerase iota incorporates dCTP opposite template G via
a G.C + Hoogsteen base pair. Structure 13, 1569–1577.
Swan, M.K., Johnson, R.E., Prakash, L., Prakash, S., and Aggarwal, A.K.
(2009a). Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase delta. Nat. Struct. Mol. Biol. 16, 979–986.
Nair, D.T., Johnson, R.E., Prakash, S., Prakash, L., and Aggarwal, A.K. (2004).
Replication by human DNA polymerase-iota occurs by Hoogsteen base-pairing. Nature 430, 377–380.
Swan, M.K., Johnson, R.E., Prakash, L., Prakash, S., and Aggarwal, A.K.
(2009b). Structure of the human Rev1-DNA-dNTP ternary complex. J. Mol.
Biol. 390, 699–709.
O’Brien, P.J., and Ellenberger, T. (2004). The Escherichia coli 3-methyladenine
DNA glycosylase AlkA has a remarkably versatile active site. J. Biol. Chem.
279, 26876–26884.
Trewick, S.C., Henshaw, T.F., Hausinger, R.P., Lindahl, T., and Sedgwick, B.
(2002). Oxidative demethylation by Escherichia coli AlkB directly reverts
DNA base damage. Nature 419, 174–178.
Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved 831
Structure
Bypass of Methylation Damage Lesions by Dpo4
Trincao, J., Johnson, R.E., Wolfle, W.T., Escalante, C.R., Prakash, S., Prakash,
L., and Aggarwal, A.K. (2004). Dpo4 is hindered in extending a G.T mismatch
by a reverse wobble. Nat. Struct. Mol. Biol. 11, 457–462.
Yang, W., and Woodgate, R. (2007). What a difference a decade makes:
insights into translesion DNA synthesis. Proc. Natl. Acad. Sci. USA 104,
15591–15598.
Vaisman, A., Ling, H., Woodgate, R., and Yang, W. (2005). Fidelity of Dpo4:
effect of metal ions, nucleotide selection and pyrophosphorolysis. EMBO J.
24, 2957–2967.
Yang, C.G., Yi, C., Duguid, E.M., Sullivan, C.T., Jian, X., Rice, P.A., and He, C.
(2008). Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound
to dsDNA. Nature 452, 961–965.
Wahl, M.C., and Sundaralingam, M. (1997). C-H.O hydrogen bonding in
biology. Trends Biochem. Sci. 22, 97–102.
Yu, B., and Hunt, J.F. (2009). Enzymological and structural studies of the
mechanism of promiscuous substrate recognition by the oxidative DNA repair
enzyme AlkB. Proc. Natl. Acad. Sci. USA 106, 14315–14320.
Waters, L.S., Minesinger, B.K., Wiltrout, M.E., D’Souza, S., Woodruff, R.V., and
Walker, G.C. (2009). Eukaryotic translesion polymerases and their roles and
regulation in DNA damage tolerance. Microbiol. Mol. Biol. Rev. 73, 134–154.
Yu, B., Edstrom, W.C., Benach, J., Hamuro, Y., Weber, P.C., Gibney, B.R., and
Hunt, J.F. (2006). Crystal structures of catalytic complexes of the oxidative
DNA/RNA repair enzyme AlkB. Nature 439, 879–884.
Westbye, M.P., Feyzi, E., Aas, P.A., Vagbo, C.B., Talstad, V.A., Kavli, B.,
Hagen, L., Sundheim, O., Akbari, M., Liabakk, N.B., et al. (2008). Human
AlkB homolog 1 is a mitochondrial protein that demethylates 3-methylcytosine
in DNA and RNA. J. Biol. Chem. 283, 25046–25056.
Zhang, H., Eoff, R.L., Kozekov, I.D., Rizzo, C.J., Egli, M., and Guengerich, F.P.
(2009). Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in
translesion synthesis past bulky N2-alkylguanine adducts. J. Biol. Chem. 284,
3563–3576.
832 Structure 19, 821–832, June 8, 2011 ª2011 Elsevier Ltd All rights reserved