Angewandte
Chemie
To gain insight into the capability of reading and extend-
ing beyond N2,3-eG by polymerases, Dpo4 was characterized
in terms of its ability to catalyze full-length extension
reactions. Sequences of the products were determined and
relative yields were estimated (summarized in Table 2 and
Table S3 of Supporting Information) from LC-MS/MS results
Table 2: Products of the extension of template–primer complexes by
Dpo4.[a]
3’-CCCCCGAGCATTCCTAAGXTACT
5’-GGGGGCTCGTAAGGATUC
Yield [%]
Base added
Figure 2. Crystal structures of Dpo4·N2,3-eG-DNA complexes (Z=C in
CCATGA
CCATGAA
CTATGA
CTATGAA
CAATGA
CGATGA
CATGA
45
7
35
8
4
<1
1
C
T
the template). a) Ternary complex of Dpo4 with dCTP and N2,3-eG-
containing duplex DNA, (Dpo4-1) and b) the orientation of the bases
with proposed hydrogen-bonding mechanism (distances shown in ꢀ).
c) Binary complex of Dpo4 with ddT across from N2,3-eG in the DNA
duplex (Dpo4-3) and d) the orientation of the bases with proposed
hydrogen-bonding mechanism. The quality of the data is demonstrated
using non-biased omit electron density maps, displayed as red mesh,
at 1.0 s in (a) and (c). Colors of the atoms: O, red; N, blue; P, orange;
F, gray.
X: 2’-F-N2,3-e-dG
A
G
deletion
C
C
2’-F-dG
dG
CCATGA
CCATGA
100
100
[a] X is 2’-F-N2,3-e-2’-deoxyarabino-guanosine (2’-F-N2,3-e-dG), 2’-fluoro-
2’-deoxyarabinoguanosine (2’-F-dG), or (unmodified) 2’-deoxyguanosine
(dG). Mass spectrometry data used to derive these results are presented
in Figures S2–S7 and Tables S4–S16 of Supporting Information.
interaction with eG. A Watson–Crick-like configuration was
seen for N2,3-eG:C base pairing (Figure 2b), whereas N2,3-
eG:T mispairing resembles a sheared base pair (Figure 2d).
Interestingly, Singer et al.[13] had suggested “wobble” pairing
but of a very different type.
(Tables S4–S17 and Figures S2–S7 of Supporting Informa-
tion). The primer was readily extended by Dpo4, bypassing
N2,3-e-dG, similar to that seen for 2’-F-dG and unmodified
G templates. With the T-containing template (3ꢀ-eGT-5ꢀ; Z =
T from Figure 1), Dpo4 produced a higher yield of extension
products with C incorporated opposite the lesion (52%, Table
2) compared to T (43%, Table 2). Similar results were seen
with the C-containing template (3ꢀ-eGC-5ꢀ; Z = C from
Figure 1) shown in Table S3 of the Supporting Information.
Thus, the insertion of T opposite N2,3-eG underscores the
mutagenic potential of this lesion. A general trend of
T misinsertion observed for the five polymerases studied
herein is in concert with reports by Singer et al. for catalysis
by AMV reverse transcriptase in a polyribo(GC) template
containing N2,3-eG,[13] but the results (pairing with C > T> A)
are at considerable variance with model calculations.[14]
To understand the base-pairing mechanisms of N2,3-eG
with C and Tresidues (see above), we determined crystal
structures of two ternary complexes Dpo4·DNA·dCTP
(Dpo4-1, 3ꢀ-eGC-5ꢀ; Dpo4-2, 3ꢀ-eGT-5) at 2.3 ꢁ resolution
and a binary complex of Dpo4·DNA (Dpo4-3, with ddT
opposite N2,3-eG) at 3.5 ꢁ resolution (Figure 2, refinement
statistics summarized in Table S17 of Supporting Informa-
tion). The active sites of all three structures resemble the
reported configuration of the so-called “type I” Dpo4–DNA
complex,[17b] where one base pair is accommodated at the
active site, and the 5’ base in the template is rotated over 908
away (Figure 2a,c and Figure S8 of Supporting Information).
Base pairing of N2,3-eG with dCTP in ternary complexes
(templates: 3’-eGC-5’ and 3’-eGT-5’) showed both N2,3-eG
and dCTP in an anti conformation. Interestingly, electron
density suggested that the G residue 3’ to the lesion is most
likely in the syn conformation to form a better stacking
Significant differences in the replication patterns and
mechanisms exist when comparing current results to our
previously reported 1,N2-eG, an isomer of N2,3-eG formed
through similar pathways.[6b] Differences in catalytic efficien-
cies and miscoding frequencies for the two lesions are
summarized in Table S18 of the Supporting Information.
Overall, 1,N2-eG has a much higher miscoding potential, with
potential base pairing with A, T, or G by different pols.[17c,18]
Extension beyond 1,N2-eG by Dpo4 yields mainly deletion
products (À1 and À2),[17c] whereas these were rare for N2,3-eG
(approximately 1%). Crystal structures of Dpo4 with 1,N2-eG
resemble “type II” structures,[17b] where the 5’ base in the
template is oriented in the active site to pair with the
incoming nucleotide, which explains the deletion products
observed in primer extension reactions.
In summary, we have successfully used a non-classical
isostere approach to stabilize an important, labile DNA
lesion, N2,3-eG.[19] Kinetic assays using representative DNA
polymerases allow quantitative assessment of the miscoding
tendency of this lesion and underscore the diversity of
biological effects that can result from isomeric DNA adducts.
Structural insights reveal the base-pairing mechanisms of the
correct base C and miscoded base T with one of the DNA
polymerases (Dpo4). The most common mispairing is con-
sistent with the reported GC to AT transition mutations
observed in the second base of codon 13 of the K-ras gene in
five out of six human VC-induced angiosarcomas,[9,20] which
are not explained by known C or G adducts (3,N4-etheno-
cytosine, N7-(2-oxoethyl)G, or 1,N2-eG).[8f,18,19,21] Thus, this
adduct (N2,3-eG) may be more relevant to the VC-induced
tumors, and its presence in unexposed humans may be an
issue in disease, in that the misincorporation patterns (N2,3-
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!
Zhao, Linlin
