N2-Substituted 2′-Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ

Article abstract of DOI:10.1002/anie.201611607

N²-Alkyl-2′-deoxyguanosine triphosphate (N²-alkyl-dGTP) derivatives with methyl, butyl, benzyl, or 4-ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N²-Benzyl-dGTP was equal to

Full text of DOI:10.1002/anie.201611607

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Communications
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International Edition: DOI: 10.1002/anie.201611607
German Edition: DOI: 10.1002/ange.201611607
Nucleotide Analogues
N2-Substituted 2’-Deoxyguanosine Triphosphate Derivatives as
Selective Substrates for Human DNA Polymerase k
A. S. Prakasha Gowda, Marietta Lee, and Thomas E. Spratt*
Abstract: N2-Alkyl-2’-deoxyguanosine triphosphate (N2-alkyl-
dGTP) derivatives with methyl, butyl, benzyl, or 4-ethynyl-
benzyl substituents were prepared and tested as substrates for
human DNA polymerases. N2-Benzyl-dGTP was equal to
dGTP as a substrate for DNA polymerase k (pol k), but was
a poor substrate for pols b, d, h, i, or n. In vivo reactivity was
evaluated through incubation of N2-4-ethynylbenzyl-dG with
wild-type and pol k deficient mouse embryonic fibroblasts.
CuAAC reaction with 5(6)-FAM-azide demonstrated that only
cells containing pol k were able to incorporate N2-4-ethynyl-
benzyl-dG into the nucleus. This is the first instance of a Y-
family-polymerase-specific dNTP, and this method could be
used to probe the activity of pol k in vivo.
polymerases were utilized to create a modified nucleotide
that can recognize a carcinogen-modified DNA template,[8]
and size-expanded dNTPs (dxNTPs) have been shown to
have some selectivity for human DNA polymerase q.[9] In this
work, we utilized the known reactivity of DNA polymerase k
to rationally design N2-benzyl-dGTPs that are highly specific
substrates for pol k. These triphosphates are the first reported
nucleotide triphosphates that are highly selective substrates
for a human Y-family polymerase. These compounds can be
utilized to probe the reactivity of pol k in vivo, and could
potentially be modified to be selective inhibitors of pol k.
The mammalian cell utilizes sixteen DNA polymerases to
replicate DNA: the four high-fidelity enzymes that duplicate
the bulk of genomic and mitochondrial DNA, together with
specialized DNA polymerases that perform roles in the DNA
damage response. Translesion DNA synthesis (TLS) poly-
merases are a subset of the specialized polymerases that are
involved in the bypass of DNA damage.[10] TLS polymerases
include the Y-family DNA polymerases, pol h, pol i, pol k,
and REV1; the B-family pol z;[10b] and perhaps other pols
such as l, n, q, and PrimPol.[11] These polymerases have
unique DNA binding sites that enable the polymerases to
bypass a variety of DNA damage. However, polymerases that
participate in lesion bypass also perform other functions. For
example, while DNA polymerase k (pol k) is the most active
polymerase in the accurate bypass of bulky N2-dG adducts,[12]
pol k also bypasses the structurally divergent 8-oxo-dG,[13]
participates in nucleotide excision repair (NER),[14] and
replicates non-B-DNA sequences,[15] and its polymerase
activity is involved in initiation of the ATR checkpoint
signal.[16] In addition, abnormal expression of pol k correlates
with increased mutations in tumors.[17] Further complicating
the matter, many of these function are not unique to pol k,
since both pol i and pol h[18] can bypass N2-dG adducts, pol d is
the major NER polymerase, and pol h can also replicate non-
B-DNA sequences.[19]
Synthetic nucleotide analogues are widely used tools in
chemical biology, diagnostics, and therapeutics. Modifications
to the Watson–Crick hydrogen-bonding face have been
employed to probe the value of Watson–Crick hydrogen
bonds in DNA replication[1] and create unnatural base pairs.[2]
Minor-groove modifications are used to elucidate critical
protein–DNA interactions,[3] while major-groove modifica-
tions have proved to be useful in exploring polymerase
enzymology[4] and cellular reactivity.[5] Inhibitors of viral
reverse transcriptases are in clinical use for treating HIV,
hepatitis B, and hepatitus C.[6] Inhibitors of human DNA
polymerase are also in use for cancer chemotherapy. Gemci-
tabine, a cytidine analogue that is incorporated into the DNA
but then inhibits DNA synthesis, is used to treat pancreatic
cancer, non-small cell lung cancer, breast cancer, and bladder
cancer.[6c] Gemcitabine is effective because it affects rapidly
growing tumor cells more than normal tissue. More recently,
specialized polymerases that are overexpressed in tumors
have been the target of inhibition studies.[7] The identification
of nucleotide analogues that are selective substrates or
inhibitors for specific polymerases is challenging because all
polymerases utilize the four canonical dNTPs, and correct
base pairing is mostly dependent on the polymerase recog-
nizing the Watson–Crick geometry. Recently, engineered
The roles of individual polymerases in the cell are difficult
to resolve, in part, because all polymerases utilize undamaged
DNA and the four dNTPs as substrates. To help elucidate the
many roles of pol k, we have designed a triphosphate that is
a highly specific substrate for pol k. Herein, we describe the
synthesis of N2-benzyl-dGTP (N2-Bn-dGTP) and show that it
is a substrate for purified pol k but a very poor substrate for
pols b, d, h, i, and n. We also show that N2-p-ethynylbenzyl-dG
(EBndG), when applied to cells, is incorporated into the
DNA only in the presence of pol k.
[*] Dr. A. S. P. Gowda, Prof. T. E. Spratt
Department of Biochemistry and Molecular Biology
Pennsylvania State University
500 University Dr., Hershey, PA 17033 (USA)
E-mail: tes13@psu.edu
Prof. M. Lee
Department of Biochemistry and Molecular Biology
New York Medical College, Valhalla, NY 10595 (USA)
The rationale for the design of a dNTP substrate specific
to pol k is illustrated in Figure 1. Pol k bypasses hydrophobic
N2-alkyl-dG adducts ranging from methyl to (benzo[a]pyren-
6-yl)methyl,[20] as well as the carcinogenic N2-7,8,9-trihy-
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201611607.
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Figure 2. Comparison of the reactivity of pols k, h, and i with N2-Bn-
dGTP. In the primer-extension assay, DNA (10 nm) and the polymerase
(100 nm) were reacted with 5 mm dGTP (a, c, e) or N2-Bn-dGTP (b, d,
f) at concentrations of 5 mm (b), or 25 mm (d, f) for the indicated
amount of time. The substrates and products were separated by
polyacrylamide gel electrophoresis (PAGE). The lower bands on the
polyacrylamide gels are the 15-mer starting material, while the upper
bands are the 16-mer products.
Figure 1. Rationale for the design of a dNTP substrate that is specific
for pol k. a) Incorporation of dCTP opposite N2-BP-dG. b) Incorpora-
tion of N2-Bn-dGTP opposite dC.
droxy-7,8,9,10-tetrahydrobenzo[a]pyren-10-yl-dG
(N2-BP-
dG; Figure 1a), with kinetic parameters approaching those
of undamaged DNA.[21] While high-fidelity polymerases
utilize interactions with the minor groove of the DNA to
enhance fidelity, pol k has an opening on the minor-groove
side of the DNA that can accommodate bulky alkyl groups
attached to the N2-position of dG in the template base.[22] We
hypothesized that this binding pocket could also accommo-
date N2-alkyl groups when bound to the incoming dGTP
(Figure 1b). We investigated the specificity of the dNTPs by
examining the in vitro reactivity with an array of human
polymerases that includes pols k, b, d, h, i, and n. These
enzymes include members of the A-, B-, X-, and Y-family
polymerases.
The in vitro activities of purified human polymerases with
N2-alkyl-dGTPs were analyzed by primer-extension assays, as
illustrated in Figure 2. The panels on the left show that pols k,
h and i readily insert dGTP opposite dC. The panels on the
right show that only pol k rapidly incorporates N2-Bn-dGTP.
Even at a higher N2-Bn-dGTP concentrations and longer time
periods, pols h and i were much less efficient at utilizing N2-
Bn-dGTP. For representative gel images of all the polymerase
reactions examined, see Figure S1 in the Supporting Infor-
mation. The in vitro reactivities of six purified polymerases
were examined as described in the Supporting Information.
The kinetic parameters are presented in Table S1 in the
Supporting Information and the reactivities are shown in
Figure 3.
Pol k reacted rapidly with the three dGTP analogues
(Figures S2,S3). The methyl and benzyl substitutions did not
impact the kpol value, while the Kd rose by a factor of two. N2-
butyl-dGTP was a slightly poorer substrate than dGTP,
leading to a 20-fold decrease in kpol. The Y-family pols h and
i, while they were not affected by the methyl substitutions,
experienced a 10000-fold reduction in kpol with the butyl
substitution (Figures S4–S7). Pol i reacted poorly with N2-Bn-
dGTP, and the reactivity of pol h further decreased due to
a decrease in kpol and an increase in Kd. In spite of the ability
of pol h to bypass N2-(2-naphthyl)methyl-dG in the template,
the polymerase is unable to utilize N2-Bu-dGP or N2-Bn-
dGTP as substrates. Of the Y-family polymerases, only pol k
Figure 3. Reactivity of dGTP (H), N2-methyl-dGTP (Me), N2-butyl-dGTP
(Bu), and N2-Bn-dGTP (Bn) with DNA polymerases k, i, h, b, n, and d/
PCNA.
reacts with N2-Bn-dGTP at rates similar to that with
unmodified dNTPs.
Next, we examined the reactivity with representatives of
the A-, B- and X-family polymerases. Humans have three A-
family polymerases: the high-fidelity pol g, which is respon-
sible for mitochondrial replication, and the low-fidelity pols q
and n. Pol q plays a role in alternative non-homologous end
joining (NHEJ), while pol n is implicated in the repair of
intrastrand crosslinks and translesion DNA synthesis. Despite
the fact that pol n shows low fidelity, it incorporated the N2-
alkyl-dGTPs very poorly (Figure S10, S11). The kpol/Kd for
N2-Bn-dGTP is reduced more than 105 -fold compared to
dGTP. The reactivity of N2-Bn-dGTP with pols g and q still
needs to be determined. Humans have four B-family poly-
merases: a, d, and e are involved in high-fidelity DNA
replication, while pol z is involved in TLS. Pol d is a B-family
polymerase that is implicated in lagging-strand replication.
We examined the reactivity of pol d/PCNAunder steady-state
conditions (Figure S12). The reaction is sensitive to small
modifications, as demonstrated by the 300-fold decrease in
kcat/Km for N2-Me-dGTP relative to dGTP (Table S1).
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Increasing the size of the substitution further decreased
reactivity, with a 105-fold decrease in kcat/Km for pol d with N2-
Bn-dGTP. Similarities among B-family polymerases suggest
that both pol a and pol e will be equally poor at utilizing N2-
Bn-dGTP as a substrate. It was previously shown that N2-
butylphenyl-dGTP was not a substrate for pols a, g, or b, but
was an inhibitor of pol a.[23] Humans have three X-family
polymerases: b, l, and m. Pol b is the primary polymerase
involved in base excision repair, while pols l and m are
involved in NHEJ. The preferred substrate for pol b is
a duplex DNA with a single-nucleotide gap. Pol b was very
inefficient at incorporating nucleotides opposite N2-BP-dG.[24]
Unsurprisingly, we found that pol b did not accommodate
steric bulk at the N2-position of the dNTP (Figures S8, S9).
Methyl substitution reduced the kpol/Kd values by over 1000-
fold. Further increases in the size of the alkyl group lead to
a decrease in kpol/Kd of more than six orders of magnitude.
The fidelity of pol k with N2-Bn-dGTP is slightly better
than with dGTP. We investigated the fidelity of the incorpo-
ration of N2-Bn-dGTP opposite dA, dC, dG, and dT as the
template base with Michaelis–Menten kinetics. As summar-
ized in Table S2, the preference for dC is higher with N2-Bn-
dGTP than with dGTP. The increased selectivity for dC as
a template is due to a reduced rate of mispair formation. The
finc value for dGTP was 0.07 for each nucleotide, while finc
averaged 0.015 for N2-Bn-dGTP.
Figure 4. Detection of EBdG in the nuclei of pol k containing cells.
Wild-type (a–c) and POLK(À/À) (d–f) cells were treated with 10 mm
EBdG for 24 h. The DNA is visualized with DAPI-staining (a,d) and
EBdG incorporation is visualized by click reaction with FAM-N3 (b,e).
Scale bars: 40 mm. g, The levels of FAM–EBdG conjugate in nuclei of
wild-type MEF [POLK(+/+); black] and POLK(À/À) (red) cells are
shown. The incubation with EBdG was performed three times. The
total number of cells are shown in violin plots for wild-type (black) and
POLK(À/À)(red) cells.
The in vitro reactions indicated that N2-Bn-dGTP is
a significantly better substrate for pol k, than for pols b, d,
h, i, or n. To determine whether this reaction occurs in vivo,
we synthesized N2-(4-ethynylbenzyl)-dG (EBdG) as de-
scribed in the Supporting Information. Attachment of the
ethynyl moiety to the benzyl group enables the use of
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) to
attach a fluorescent group to the nucleoside and thus
determine the amount and location of EBdG in the cell.[5]
Labelling of the nuclei is shown in Figure 4a–c, in which
mouse embryonic fibroblasts (MEFs) were incubated with
10 mm EBdG for 24 h. The cells were fixed and the ethynyl
groups were reacted with 5(6)-FAM-azide, and the nuclei
were identified by 4’,6-diamidino-2-phenylindole (DAPI)
staining of the DNA. The complete overlap of the DAPI
(Figure 4a) and FAM (Figure 4b) signals indicates that the
cells incorporated EBdG into the nucleus.
addition, incorporation into the DNA is dependent on the
presence of pol k.
In conclusion, we have found that N2-alkyl-dGTPs are
substrates for DNA pol k. The kinetic parameters with N2-Bn-
dGTP are only slightly slower than with dGTP. The fidelity of
the pol k catalyzed incorporation is slightly better for N2-Bn-
dGTP than it is for dGTP. We also found that N2-Bn-dGTP
reacted slowly with representatives from the A-family (n), B-
family (d), or an X-family (b) polymerases; the kpol/Kd or kcat
/
To determine whether pol k is the polymerase that
incorporates EBdG into DNA in mammalian cells, we
incubated MEF cells deficient in pol k with the nucleoside
and then analyzed incorporation by visualization after the
click reaction. Figure 4d–f shows that the pol k deficient MEF
cells were unable to incorporate EBdG into the nucleus. The
relative intensities of FAM signal and the DAPI signal were
measured for each nucleus. The first lane of Figure 4g shows
the background level of FAM fluorescence in the nucleus. At
10 nm EBdG, approximately half of the wild-type cells
incorporated a small amount of the nucleoside. Above this
concentration, all cells incorporated EBdG. In cells without
pol k, EBdG is not incorporated into the DNA. These results
indicate that EBdG diffuses into the nucleus, is converted into
the triphosphate, and is incorporated into the DNA. In
Km values are 105 times lower that of pol k. We also found that
the ethynyl nucleoside N2-EBn-dG is incorporated into DNA
in cells by pol k. These reagents could prove very useful for
elucidating the cellular activities of pol k, and could from the
basis for selective inhibition of pol k.
Acknowledgements
This work was supported by the National Institute of
Environmental Health Sciences (ES 021762 and ES 014737).
We thank Cyrus Vasiri for the gift of wild-type and
POLK(À/À) MEF cells.
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Conflict of interest
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a provisional patent on the compounds.
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Manuscript received: November 28, 2016
Revised: January 12, 2017
Final Article published: && &&, &&&&
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Nucleotide Analogues
Heading for the pols: N2-Alkyl-2’-deoxy-
guanosine triphosphate (N2-alkyl-dGTP)
derivatives with methyl, butyl, benzyl, 4-
ethynylbenzyl substituents were prepared
and tested as substrates for human DNA
polymerases. N2-Benzyl-dGTP was equal
to dGTP as a substrate for DNA poly-
merase k (pol k), but was a poor sub-
strate for pols b, d, h, i, or n. In vivo
reactivity was evaluated, and only cells
containing pol k were able to incorporate
N2-4-ethynylbenzyl-dG into the nucleus.
A. S. P. Gowda, M. Lee,
T. E. Spratt*
&&& — &&&
N2-Substituted 2’-Deoxyguanosine
Triphosphate Derivatives as Selective
Substrates for Human DNA Polymerase k
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