Biotinylated lithocholic acids for affinity chromatography of mammalian DNA polymerases α and β

Article abstract of DOI:10.1016/S0960-894X(01)00725-9

Biotinylated lithocholic acids have been synthesized. The compounds inhibited mammalian DNA polymerases α and β with dose-dependent manner. The streptavidine columns conjugated with the synthetic biotinylated compounds chromatographed both two enzymes eluted by KCl solution at the different concentrations.

Full text of DOI:10.1016/S0960-894X(01)00725-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 287–290
Biotinylated Lithocholic Acids for Affinity Chromatography of
Mammalian DNA Polymerases ꢀ and ꢁ
a
a
b
b
Madoka Watanabe, Shinya Hanashima, Yoshiyuki Mizushina, Hiromi Yoshida,
a
a
a,
Masahiko Oshige, Kengo Sakaguchi and Fumio Sugawara *
aDepartment of Applied Biological Science, Frontier Research Center for Genomic Drug Discovery, Tokyo University of Science,
Noda, Chiba 278-8510, Japan
b
Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, High Technology Research Center,
Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
Received 6July 2001; accepted 26October 2001
Abstract—Biotinylated lithocholic acids have been synthesized. The compounds inhibited mammalian DNA polymerases a and b
with dose-dependent manner. The streptavidine columns conjugated with the synthetic biotinylated compounds chromatographed
both two enzymes eluted by KCl solution at the different concentrations. # 2002 Elsevier Science Ltd. All rights reserved.
Lithocholic acid (LCA, 1), one of secondary bile acids,
is known to promote tumorigenesis in rats induced by
the monoalkylating agent, N-methyl-N-nitro-N-nitroso-
revealed that eukaryotic cells contain at least twelve types
of DNA pol. From this reason, we aimed to synthesize
biotinylated LCAs and develop affinity column chroma-
tography for further biochemical research of DNA pol.
1
,2
guanidine.
LCA inhibits the activity of eukaryotic
DNA polymerase (pol) b, which is known to be
involved in DNA excision repair, and DNA pol a, which
3
roles DNA replication process in cell cycle. The IC
value of DNA pol a is less than that of DNA pol b
Fig. 1). These observations suggest the possible
5
0
Synthesis of Biotinylated LCAs
(
Benzyl ester (2) of LCA (1) was prepared from the reaction
of benzyl bromide and DBU in benzene, then 3-hydroxyl
was protected with the treatment of CBZ b-alanine with
EDCI/DMAP in CH Cl to give 3. After debenzylation
mechanisms for tumorigenesis by LCA. Recently,
Mizushina et al. indicated that LCA binds the template-
primer binding domain (8 kDa) but not to the catalytic
2
2
1
15
domain (31 kDa) of DNA pol b. On H– N HMQC
NMR experiment of DNA pol b associated with LCA,
the 8 kDa domain bound to LCA as a 1:1 complex.
with Pd(OH) under hydrogen atmosphere, the resulting
2
amine (4) was treated with N-hydroxysuccinimide-biotin,
biotinamidocaproate N-hydroxysuccinimide ester and 6-
(biotinamidecaproylamide)caproic acid N-succinimide
ester in DMF in the presence of Et N to give 5, 6 and 7,
3
respectively (Scheme 1).
4
Three amino acid residues (Lys60, Leu77 and Thr79) in
this domain were mainly interacted with LCA. It is
important to isolate and use the specific inhibitors of
DNA pol as molecular probes to understand not only
in vivo functions but also precise biological cross talks
in cell cycle. It is also mentioned that the DNA pol
inhibitors can be useful for cancer chemotherapy with
5
DNA targeting drugs such as bleomycin.
The isolations of DNA pols by using many types of col-
umn chromatography (e.g., phoshocellulose, human serum
conjugated sepharose, rabbit IgG conjugated sepharose
and then monoclonal antibody conjugated sepharose col-
umn chromatography for DNA pol b) are still laborious
and time-consuming works, because recent researches have
*Corresponding author. Tel.:+81-471-24-1501; fax:+81-471-23-9767;
e-mail: sugawara@rs.noda.sut.ac.jp
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00725-9

288
M. Watanabe et al. / Bioorg. Med. Chem. Lett. 12 (2002) 287–290
(Table 1). In the kinetic analyses, poly(dA)/oligo(dT)1
2ꢀ18
and dTTP were used as the DNA template-primer and
nucleotide substrate, respectively. Double reciprocal
plots of the results indicated that the LCA-mediated
inhibition of the DNA pol a activity was non-competitive
with both the DNA template-primer and the nucleotide
substrate. In the case of the DNA template-primer, 56.3
and 31.8% decreases in maximum velocity (Vmax) were
observed in the presence of 25 and 50 pmol/h LCA,
respectively, whereas the apparent Michaelis constant
(
K ) was unchanged at 13.0 mM. The Km value for the
m
nucleotide substrate was 1.65 mM, and the Vmax value
for the nucleotide substrate decreased from 29.2 to 14.8
pmol/h in the presence of 50 mM LCA. The inhibition
constant (K ) value for DNA pol a, obtained from
i
Scheme 1. Reagents and conditions: (a) benzyl bromide, DBU, ben-
zene reflux, 16h (75.2%); (b) CBZ- b-alanine, EDCI, DMAP, dry
Dixon plots, was found to be 15.3 and 22.5 mM for the
DNA template-primer and nucleotide substrate, respec-
tively. The affinity of LCA might be higher at the DNA
template-primer binding site than at the nucleotide
substrate-binding site. On the other hand, the DNA
pol b inhibition by LCA was competitive with both
the DNA template-primer and the nucleotide substrate,
because the Vmax value was unchanged at a concentra-
tion of 111.0 and 62.5 pmol/h, respectively. The Ki
values for DNA template-primer and nucleotide sub-
strate were 4.70 and 6.62 mM, respectively. These data
suggested that the binding modes of LCA to DNA pol a
and DNA pol b are different from each other. As shown
in Figure 1, the biotinylated LCA could inhibit the
DNA pol a and b activity. The inhibition of DNA pol a
activity by compound 7 was non-competitive for both
the DNA template-primer (the Km was unchanged at
13.0 mM) and the nucleotide substrate (the Km was
unchanged at 1.65 mM), and the inhibition of DNA
pol b activity by compound 7 was competitive for both
the DNA template-primer (the Vmax was unchanged at
111 pmol/h) and the nucleotide substrate (the Vmax was
unchanged at 62.5 pmol/h). The inhibition mode for
DNA pol a and b by biotinylated LCA was the same as
that by LCA.
CH
2
Cl
82.0%); (d) N-hydroxysuccinimide-biotin, DMF, Et
e) biotinamidocapronate N-hydroxysuccinimide ester, DMF, Et
69.2%); (f) 6-(biotinamidecaproylamide)caproic acid N-succinimide
N, 5 h (77.5%).
2
, 16h (99.0%); (c) Pd(OH)
2
, H
2
, EtOAc/EtOH/H
N, 5 h (81.8%);
N, 5 h
2
O (10:1:1), 5 h
(
(
(
3
3
ester, DMF, Et
3
Effects of biotinylated lithocholic acids on the activities
of mammalian DNA polymerases ꢀ and ꢁ
Figure 1 shows the inhibitory dose–response curves of
LCA (1) and the biotinylated compounds (5–7) against
the mammalian DNA pol a and b. All of these com-
pounds dose-dependently inhibited the activities of calf
DNA pol a and rat DNA pol b. LCA strongly inhibited
the activity of DNA pol b rather than that of DNA pol
a. On the other hand, the inhibitory dose curves for
DNA pols a and b by 5 and 6 were almost similar, and 7
inhibited DNA pol a more effectively. The IC50 values
of DNA pol a activity by LCA (1), compound 5, 6, and
7
were 40, 29, 42 and 10 mM, respectively (Fig. 1A). The
IC50 values of pol b activity by 1, 5, 6 and 7 were 11, 31,
6and 32 mM, respectively (Fig. 1B).
4
Mode of inhibition of DNA polymerase ꢀ and ꢁ by
LCA and compound 7
Interaction of LCA and DNA polymerases ꢀ and ꢁ
To elucidate the inhibitory mechanism, the extent of
inhibition was studied as a function of the DNA tem-
plate-primer or nucleotide substrate concentrations
DNA pol a and b were charged to the biotinylated
LCA-streptavidine columns and eluted by KCl solution.
Figure 1. Dose–response curves of LCA (1) and the biotinylated compounds (5, 6, 7). The enzymes used (0.05 units each) were calf DNA pol a and
rat DNA pol b. The DNA polymerase activities were measured as described in the text. DNA polymerase activity in the absence of compound was
taken as 100%.

M. Watanabe et al. / Bioorg. Med. Chem. Lett. 12 (2002) 287–290
289
Table 1. Kinetic analysis of the inhibition by lithocholic acid 1 and the biotinylated compound 7 on the activities of DNA pol, as a function of the
DNA template-primer dose and the nucleotide substrate concentration
Compd
Enzyme
0.05 units each)
Substrate
Compd concn
(mM)
K
(mM)
m
V
max
(pmol/h)
K
(mM)
i
Inhibition mode
(
1
Pol a
Pol b
Template-primera
0
55.6
31.3
17.7
29.2
24.9
14.8
2
5
0
0
5
0
13.00
15.3
Non-competitive
Non-competitive
Competitive
5
Nucleotide substrateb
Template-primera
2
5
1.65
22.5
4.7
5
0
0
5
0
9.41
18.3
3.05
6.28
7.86
111.0
62.5
1
Nucleotide substrateb
6.6
Competitive
1
7
Pol a
Pol b
Template-primera
Nucleotide substrateb
Template-primera
0
5
0
0
5
0
0
0
0
0
0
0
55.6
33.5
21.3
29.2
24.3
17.8
13.00
1.65
7.7
5.5
Non-competitive
Non-competitive
Competitive
1
1
6.47
9.98
21.70
3.05
6.67
9.49
1
2
111.0
62.5
12.8
20.1
Nucleotide substrateb
1
2
Competitive
a
Poly(dA)/oligo(dT)12ꢀ18 (=2/1).
dTTP.
b
In the columns conjugated with the compounds 5, 6 and
, the activities of DNA pol a were appeared at 430, 450
and 500 mM of KCl concentrations, respectively. The
cal shifts were expressed by d ppm from TMS as inter-
nal standard. Optical rotation values were determined
on JASCO polarimeter P1010.
7
activities of DNA pol b were observed at 400, 370 and
1
3
40 mM KCl concentrations from the streptavidine
Compound 5: C H N O S. H NMR (CD OD): LCA
3
3
7
59
2
6
sepharose columns conjugated with the compounds 5, 6,
and 7, respectively. Longer linker between LCA and
biotin tended to elute by low concentration of KCl.
Thus, compound 7 was the best candidate for DNA pol
affinity chromatography in the tested compounds. The
streptavidine column chromatography by using com-
pound 7 is able to isolate DNA pol a and b from the
mixture (Fig. 2). The high incorporation of dTTP for
activated DNA indicated the enzymatic activity of
polymerase b and the high incorporation of poly(dA)/
oligo(dT) implied the activity of DNA pol a. Thus, the
isolation of the mixture of DNA pols a and b was
enabled on the affinity column chromatography used by
the derivative of inhibitor. In addition to this, the col-
umn may be possible to isolate currently unknown
enzymes regarding to DNA replication and/or repair
process in the cell cycle.
unit d 0.69 (3H, s), 0.94 (3H, d, J=6.6 Hz), 0.96 (3H, s),
1.05–1.21 (6H, m), 1.26–1.34 (4H, m), 1.41–1.46 (6H,
m), 1.50–1.76(5H, m), 1.82–1.92 (3H, m), 2.03 (1H, d,
J=11.6Hz), 2.18 (2H, dd, J=6.4, 6.4 Hz), 2.19 (1H,
m), 4.76(1H, m); biotin unit: d 1.42 (2H, m), 1.61 (2H,
m), 1.70 (2H, m), 2.33 (2H, m), 2.71 (1H, d, J=12.7
Hz), 2.91 (1H, dd, J=5.0, 12.7 Hz), 3.19 (1H, m), 4.30
(1H, dd, J=4.4, 7.8 Hz), 4.48 (1H, dd, J=5.0, 7.8 Hz);
b-Ala unit: d 2.45 (2H, t, J=6.6 Hz), 3.42 (2H, J=6.6
Experimental
NMR and FABMS data
Synthetic compounds were characterized with both
NMR and FABMS. FAB mass spectral data were col-
lected on a JEOL JMS-700 with negative ion mode on
glycerol matrix and NMR data were obtained from
1
Figure 2. DNA pol a and b elution profiles from streptavidine
sepharose column conjugated with 7.
Bruker DRX-400 (400 MHz for H in CD OD. Chemi-
3

290
M. Watanabe et al. / Bioorg. Med. Chem. Lett. 12 (2002) 287–290
ꢀ
ꢁ
0
dTTP) into synthetic template-primers [i.e., poly(dA)/
oligo(dT), A/T=2/1] in 60 min at 37 C under the nor-
8,9
Hz). FABMS: m/z 672.62 ([MꢀH] ). [a] +52.8 (c
1 nmol of deoxyribonucleotide 5 -triphosphates (i.e.,
D
0
.1, MeOH).
ꢁ
mal reaction conditions for each enzyme.
1
Compound 6: C H N O S. H NMR: LCA unit d
4
3
70
4
7
0
1
1
.59 (3H, s), 0.85 (3H, d, J=6.6 Hz), 0.86 (3H, s), 1.07–
.18 (6H, m), 1.28–1.34 (4H, m), 1.41–1.52 (6H, m),
.57–1.70 (5H, m), 1.83–1.91 (3H, m), 2.03 (1H, d,
Affinity column chromatography conjugated with
biotinylated lithocholic acids
J=11.6Hz), 2.18 (2H, t, J=7.3 Hz), 2.20 (1H, m), 4.70
(
(
(
1H, m); biotin unit: d 1.43 (2H, m), 1.59 (2H, m), 1.66
2H, m), 2.20 (2H, m), 2.72 (1H, d, J=12.7 Hz), 2.91
1H, dd, J=5.0, 12.7 Hz), 3.18 (1H, m), 4.30 (1H, dd,
Each biotinylated LCA (5–7) (5 mg each) was dissolved
in 20% DMSO and mixed with 1.0 mL of streptavidine
sepharose, respectively. The purified pol a (3 units) or
pol b (2.0 mg) was loaded onto the columns equilibrated
with 50 mM Tris–HCl (pH 7.5) and 50 mM KCl buffer.
The column was washed with 8 mL of the same buffer
and then the enzymes were eluted by a 24 mL linear
gradient of KCl solutions from 50 to 950 mM in Tris–
HCl (pH 7.5) buffer. DNA pols a and b activities and
protein concentration of the fractions (0.4 mL each) were
J=4.4, 7.8 Hz), 4.48 (1H, dd, J=5.0, 7.8 Hz); b-Ala
unit: d 2.49 (2H, t, J=6.6 Hz), 3.41 (2H, t, J=6.6 Hz);
aminocaproylamide unit d 1.32 (2H, m), 1.43 (2H, m),
2
7
.17 (2H, m), 2.25 (2H, m), 3.15 (2H, m). FABMS: m/z
ꢀ
ꢁ
85.55 ([MꢀH] ). [a] +42.7 (c 0.1, MeOH).
D
1
Compound 7: C H N O S. H NMR (CD OD): LCA
3
4
9
81
5
6
8
,9
unit d 0.69 (3H, s), 0.95 (3H, d, J=6.4 Hz), 0.96 (3H, s),
.03–1.18 (6H, m), 1.28–1.37 (4H, m), 1.41–1.54 (6H,
m), 1.60–1.70 (5H, m), 1.83–1.90 (3H, m), 2.03 (1H, d,
determined by standard DNA pol assay methods and
1
0
1
the method of Bradford, respectively.
J=11.6Hz), 2.17 (3H, m), 4.70 (1H, m); biotin unit: d
1.44 (2H, m), 1.63 (2H, m), 1.71 (2H, m), 2.30 (2H, m),
2.71 (1H, d, J=12.7 Hz), 2.91 (1H, dd, J=5.0, 12.7 Hz),
3.19 (1H, m), 4.29 (1H, dd, J=4.4, 7.8 Hz), 4.48 (1H,
Acknowledgements
This work was partly supported by Inoue Research
Award for Young Scientists from Inoue Foundation for
Science (to Y.M.). This work was also supported par-
tially supported by Grants-in-Aid 12780442 (to Y.M.)
12660103 (to F.S.) from the Ministry of Education,
Science, Sports and Culture of Japan. F.S. also thanks
Uehara Memorial Foundation and The Naitoh Foun-
dation for financial support.
dd, J=5.0, 7.8 Hz); b-Ala unit: d 2.49 (2H, t, J=6.6
Hz), 3.41 (2H, t, J=6.6 Hz); aminocaproylamide unit d
1
m), 2.20–2.30 (4H, m), 3.10–3.20 (4H, m). FABMS: m/z
.28–1.32 (4H, m), 1.39–1.46(4H, m), 2.13–2.18 (4H,
ꢀ
ꢁ
8
98.67 ([MꢀH] ). [a] +5.7 (c 0.1, MeOH).
D
DNA polymerase assay
DNA pol a was purified from calf thymus by immuno-
affinity column chromatography as described pre-
6
,7
viously. Recombinant rat DNA pol b was purified
from Escherichia coli JMpb5 as described by Date et al.
References and Notes
7
1. Narisawa, T.; Magadia, N. E.; Weisburger, J. H.; Wynder,
E. L. J. Natl. Cancer Inst. 1974, 53, 1093.
2. Reddy, B. S.; Narisawa, T.; Weisburger, J. H.; Wyner, E. L.
J. Natl. Cancer Inst. 1976, 56, 441.
The reaction mixtures for DNA pols a and b were
described previously. The substrates of DNA poly-
8
,9
3
merases were used poly(dA)/oligo(dT)1
and [ H]-
2ꢀ18
3
. Ogawa, A.; Murate, T.; Suzuki, M.; Nimura, Y.; Yoshida,
S. Jpn. J. Cancer Res. 1998, 89, 1154.
. Mizushina, Y.; Ohkubo, T.; Sugawra, F.; Sakaguchi, K.
Biochemistry 2000, 39, 12606.
. Deng, J.-Z.; Stark, S. R.; Sabat, M.; Hecht, S. M. J. Nat.
dTTP as template-primer DNA and nucleotide sub-
strate, respectively. LCA and the biotinylated com-
pounds (5, 6, 7) were dissolved in DMSO at various
concentrations and sonicated for 30 s. Four mL of each
of the sonicated sample was mixed with 16 mL of each
enzyme (final 0.05 units) in 50 mM Tris–HCl (pH 7.5)
4
5
Prod. 2000, 63, 1356.
6. Tamai, K.; Kojima, K.; Hanaichi, T.; Masaki, S.; Suzuki,
M.; Umekawa, H.; Yoshida, S. Biochim. Biophys. Acta 1988,
containing 1 mM dithiothreitol, 50% glycerol and
ꢁ
9
7
50, 263.
. Date, T.; Yamaguchi, M.; Hirose, F.; Nishimoto, Y.;
0
.1 mM EDTA, and kept at 0 C for 10 min. These
inhibitor-enzyme mixtures (8 mL) were added to 16 mL
of each of the enzyme standard reaction mixtures, and
Tanihara, K.; Matsukage, A. Biochemistry 1988, 27, 2983.
8. Mizushina, Y.; Tanaka, N.; Yagi, H.; Kurosawa, T.;
Onoue, M.; Seto, H.; Horie, T.; Aoyagi, N.; Yamaoka, M.;
Matsukage, A.; Yoshida, S.; Sakaguchi, K. Biochim. Biophys.
Acta 1996, 1308, 256.
9. Mizushina, Y.; Yoshida, S.; Matsukage, A.; Sakaguchi, K.
Biochim. Biophys. Acta 1997, 1336, 509.
10. Bradford, M. M. Anal. Biochem. 1976, 2, 248.
ꢁ
incubation was carried out at 37 C for 60 min. The
activity without the inhibitor was considered 100%,
and the remaining activities at each concentration of
inhibitor were determined as percentages of this value.
One unit of each DNA pol activity was defined as the
amount of enzyme that catalyzes the incorporation of