Tài liệu Báo cáo khoa học: Ionic strength and magnesium affect the specificity of Escherichia coli and human 8-oxoguanine-DNA glycosylases pdf

Ionic strength and magnesium affect the specificity of
Escherichia coli and human 8-oxoguanine-DNA
glycosylases
Viktoriya S. Sidorenko
1
, Grigory V. Mechetin
1
, Georgy A. Nevinsky
1,2
and Dmitry O. Zharkov
1,2
1 SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
2 Department of Natural Sciences, Novosibirsk State University, Russia
In all living organisms DNA is subject to ongoing
damage by various environmental and endogenous
factors [1]. One of the most frequently encountered
base lesions is 8-oxo-7,8-dihydroguanine (8-oxoG),
produced by oxidative stress to the steady-state level
of $ 1 · 10
6
guanines in human DNA [2]. 8-oxoG is
mutagenic due to its ability to form a stable Hoog-
sten pair with A [3] and its propensity to direct the
incorporation of dAMP by DNA polymerases [4]. If
left uncorrected, the resulting 8-oxoG:A mispair is
converted to a T:A pair in the next round of replica-
tion, producing a G:C fi T:A transversion mutation,
the type frequently encountered in human cancers
[5,6].
The consequences of 8-oxoG’s appearance in DNA
are counteracted by a three-tier enzymatic ‘GO system’

[7–9], part of general base-excision repair system [10].
In bacteria, once it has emerged in DNA in the context
of a G:C pair, the 8-oxoG base is excised from the
8-oxoG:C pair by formamidopyrimidine-DNA glycosy-
lase (Fpg, EC 3.2.2.23); in eukaryotes it is excised by
8-oxoguanine-DNA glycosylase OGG1, followed by
Keywords
8-oxoguanine; DNA damage; DNA
glycosylase; DNA repair; substrate
specificity
Correspondence
D. O. Zharkov, SB RAS Institute of
Chemical Biology and Fundamental
Medicine, Novosibirsk 630090, Russia
Fax: +7 383 333 3677
Tel: +7 383 335 6226
E-mail:
(Received 20 February 2008, revised 18
April 2008, accepted 23 May 2008)
doi:10.1111/j.1742-4658.2008.06521.x
An abundant oxidative lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), often
directs the misincorporation of dAMP during replication. To prevent muta-
tions, cells possess an enzymatic system for the removal of 8-oxoG. A key
element of this system is 8-oxoguanine-DNA glycosylase (Fpg in bacteria,
OGG1 in eukaryotes), which must excise 8-oxoG from 8-oxoG:C pairs but
not from 8-oxoG:A. We investigated the influence of various factors,
including ionic strength, the presence of Mg
2+
and organic anions, poly-
amides, crowding agents and two small heterocyclic compounds (biotin

and caffeine) on the activity and opposite-base specificity of Escherichia coli
Fpg and human OGG1. The activity of both enzymes towards 8-oxoG:A
decreased sharply with increasing salt and Mg
2+
concentration, whereas
the activity on 8-oxoG:C was much more stable, resulting in higher oppo-
site-base specificity when salt and Mg
2+
were at near-physiological concen-
trations. This tendency was observed with both Cl
)
and glutamate as the
major anions in the reaction mixture. Kinetic and binding parameters for
the processing of 8-oxoG:C and 8-oxoG:A by Fpg and OGG1 were deter-
mined under several different conditions. Polyamines, crowding agents,
biotin and caffeine affected the activity and specificity of Fpg or OGG1
only marginally. We conclude that, in the intracellular environment, the
specificity of Fpg and OGG1 for 8-oxoG:C versus 8-oxoG:A is mostly due
to high ionic strength and Mg
2+
.
Abbreviations
8-oxoG, 8-oxo-7,8-dihydroguanine; AP, apurinic ⁄ apyrimidinic; KGlu, potassium glutamate; THF, tetrahydrofuran.
FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3747
repair to restore the original G:C pair. Importantly,
both Fpg and OGG1 are much less likely to excise
8-oxoG from 8-oxoG:A substrates, because if these
mispairs are generated by the incorporation of dAMP
opposite 8-oxoG, such excision would immediately fix
the G:C fi T:A transversion. Instead, 8-oxoG:A

mispairs are processed by removal of A by the DNA
glycosylases MutY (in bacteria) or MUTYH (in
eukaryotes) and conversion of 8-oxoG:A to 8-oxoG:C
in the first round of repair, followed by a Fpg- or
OGG1-initiated second round of repair. The third
member of the GO system, MutT ⁄ NUDT1 protein,
hydrolyzes 8-oxodGTP, thus preventing incorporation
of 8-oxoG into DNA during replication [7–9]. Inacti-
vation of the GO system increases the mutagenesis rate
in bacteria [11] and increases the risk of cancer devel-
opment in mouse models [12,13] and in humans
[14,15].
Discrimination in favor of 8-oxoG:C and against
8-oxoG:A mispairs by Fpg and OGG1 is a key feature
on which the GO system is built. Although Fpg and
OGG1 share no similarity in either their sequence or
structure, the crystal structures of these proteins reveal
extensive sets of bonds with the C base opposite the
lesion [16–18]. Furthermore, stopped-flow studies sug-
gest that additional discrimination of the base opposite
the lesion may occur at earlier stages of substrate bind-
ing by both enzymes [19,20].
Although published studies on the opposite-base
specificity of Fpg and OGG1 [19–26] agree that
8-oxoG:C substrates are preferred over 8-oxoG:A
substrates, the multitude of used assay systems pre-
cludes a systematic analysis of the influence that may
be exerted by various reaction factors on this specific-
ity. In most kinetic studies, the activity of DNA gly-
cosylases is assayed in well-defined systems that

include a buffer (often non-physiological, such as Tris
or Good buffers), a salt (usually NaCl or KCl) and
stabilizing agents (usually a metal chelator, a thiol
reagent and glycerol). In living cells, the reactions
catalyzed by DNA-dependent proteins may be
affected by ionic strength, the concentration of diva-
lent cations such as Mg
2+
, the nature of the buffer-
ing agents, the presence of competing polyamines and
other small molecules, and crowding by other macro-
molecules. Their effects on the specificity of 8-oxoG
excision have never been studied. Because these fac-
tors may be important for the efficiency of correct
8-oxoG repair, in this study we address how the rela-
tive efficiency of 8-oxoG excision from pairs with C
and A by Fpg and OGG1 depends on buffer compo-
sition, ionic strength, Mg
2+
concentration and several
other factors.
Results
Effects of ionic strength and divalent cations on
the activity and specificity of Fpg and OGG1
The conditions inside a living cell differ from most
buffer systems in which the activity and specificity of
Fpg and OGG1 have been studied. For example, in
Escherichia coli the intracellular concentrations of
Na
+

and Cl
)
ions are $ 5mm, the major intracellular
monovalent cation is K
+
($ 200–250 mm), free diva-
lent cations are mostly Mg
2+
($ 10 mm) and anions
are represented by a mixture of organic acids, amino
acids, inorganic phosphate and nucleic acids [27]. To
explore the dependence of the activity and specificity
of Fpg and OGG1 on general ionic strength and the
presence of divalent cations, we conducted a factorial
design experiment in which both factors were varied
by changing the concentration of KCl and MgCl
2
,
whereas the buffering agent (potassium phosphate,
KP
i
) was kept constant at 25 mm (Table 1; Series 1).
Processing of 8-oxoG:C and 8-oxoG:A substrates was
followed in a single time point assay in a linear kine-
tics range. Mechanistically, Fpg and OGG1 differ in
their ability to catalyze cleavage of the apurinic ⁄ apyri-
midinic (AP) site via elimination of its 3¢-phosphate
(AP lyase activity) after excision of the damaged base
(DNA glycosylase activity). Fpg efficiently catalyzes
b,d-elimination at the AP site so that these reactions

cannot be separated kinetically [23,28]; therefore, unas-
sisted cleavage of substrate DNA by this enzyme was
used as the assay endpoint. The AP lyase activity of
OGG1 proceeds via b-elimination and is much less effi-
cient than its glycosylase activity [25,26]. Two assay
endpoints were used in this case: glycosylase activity
was measured after full thermal degradation of the AP
site left by base excision, whereas the AP lyase activity
was assayed as unassisted cleavage of the substrate by
Table 1. Outline of the factorial design activity experiments.
Series
Varied reaction mixture
components Concentrations
1 KCl 0, 50, 100, 150, 200 m
M
MgCl
2
0, 5, 10, 15, 20 mM
2 KGlu 0, 50, 100, 150, 200 mM
MgCl
2
0, 5, 10, 15, 20 mM
KP
i
0, 25 mM
3 Spermine or spermidine 0, 1, 10, 100, 1000 lM
MgCl
2
0, 5, 10, 15, 20 mM
4 Poly(ethylene glycol)

4000 or 8000
0, 0.05, 0.1, 0.2, 0.5,
1, 2, 5%
MgCl
2
0, 5, 10, 15, 20 mM
Factors affecting the specificity of Fpg and OGG1 V. S. Sidorenko et al.
3748 FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS
the enzyme. In the experiments described here, all
these activities were assayed unless indicated otherwise.
Both enzymes showed a decrease in the efficiency of
cleavage of both substrates with increasing ionic
strength and Mg
2+
concentration (Fig. 1). However, in
all cases, the activities of both Fpg and OGG1 on
8-oxoG:A decreased much more sharply than on the
8-oxoG:C. Whereas in the absence of KCl and MgCl
2
in the reaction mixture, the activities on these sub-
strates were at least of the same order of magnitude
and the opposite-base specificity (C ⁄ A specificity,
defined as the ratio of cleavage of the 8-oxoG:C to
8-oxoG:A substrate under identical conditions) in all
cases was the lowest, an increase in both salts to near-
physiological concentrations led to an $ 10–50-fold
preference for C compared with A opposite the lesion.
The activity of Fpg on 8-oxoG:C was more sensitive to
variations in buffer composition than the activity of
OGG1 on the same substrate (compare Fig. 1A,D

and G); OGG1 was inhibited only by the highest con-
centrations of KCl and MgCl
2
. Interestingly, the AP
lyase activity of OGG1 on 8-oxoG:A at any given
0
10
20
30
40
50
0
50
100
150
200
0
5
10
15
20
[
P
]
,
n
M
K
C
l

,
m
M
M
g
C
l
2
,
m
M
0
2
4
6
8
10
12
0
50
100
150
200
0
5
10
15
20
[
P

]
,
n
M
K
C
l
,
m
M
M
g
C
l
2
,
m
M
0
10
20
30
40
50
60
0
50
100
150
200

0
5
10
15
20
C
/
A
K
C
l
,
m
M
M
g
C
l
2
,
m
M
0
10
20
30
40
0
50
100

150
200
0
5
10
15
20
[
P
]
,
n
M
KC
l
,
m
M
M
g
C
l
2
,
m
M
0
5
10
15

20
0
50
100
150
200
0
5
10
15
20
[
P
]
,
n
M
K
C
l
,
m
M
M
g
C
l
2
,
m

M
0
5
10
15
20
25
30
35
0
50
100
150
200
0
5
10
15
20
C
/
A
K
C
l
,
m
M
M
g

C
l
2
,
m
M
0
2
4
6
8
10
0
50
100
150
200
0
5
10
15
20
[
P
]
,
n
M
K
C

l
,
m
M
M
g
C
l
2
,
m
M
0
2
4
6
8
10
12
0
50
100
150
200
0
5
10
15
20
[

P
]
,
n
M
K
C
l
,
m
M
M
g
C
l
2
,
m
M
0
2
4
6
8
10
12
0
50
100
150

200
0
5
10
15
20
C
/
A
K
C
l
,
m
M
M
g
C
l
2
,
m
M
AB C
DE F
GH I
Fig. 1. Activity and specificity of Fpg and OGG1 in buffers with different concentrations of KCl and MgCl
2
. (A–C) Fpg, (D–F) glycosylase
activity of OGG1, (G–I) AP lyase activity of OGG1. The extent of cleavage of 8-oxoG:C (A, D, G) or 8-oxoG:A (B, E, H) or the C ⁄ A specificity

(C, F, I) is plotted against the concentrations of the salts. [P], product concentration. Note the different scales in (A, D, G) compared with (B,
E, F). The concentration of the enzyme and both substrates were kept constant in the analysis of each activity (see Experimental procedures
for the reaction conditions). Means of two independent experiments are shown.
V. S. Sidorenko et al. Factors affecting the specificity of Fpg and OGG1
FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3749
concentration of KCl decreased more slowly than its
DNA glycosylase activity. Overall, the highest oppo-
site-base specificity for Fpg was observed at 5–15 mm
MgCl
2
and 50–150 mm KCl (Fig. 1C), for OGG1
glycosylase activity, at 5–10 mm MgCl
2
and 150–
200 mm KCl (Fig. 1F) and for its AP lyase activity, at
10–20 mm MgCl
2
and 100–200 mm KCl (Fig. 1I).
To analyze the impact of Mg
2+
on the opposite-
base specificity of Fpg in more detail, we measured the
steady-state kinetic parameters for the cleavage of
8-oxoG:C and 8-oxoG:A by this enzyme in the pres-
ence and absence of 10 mm MgCl
2
. The concentration
of KCl in these experiments was 50 mm, because
higher ionic strengths improved Fpg specificity at the
cost of a reduction in 8-oxoG:A cleavage to very low

levels, making the determination of individual kinetic
constants problematic. The C ⁄ A preference measured
in the factorial design experiments under these con-
ditions was 5.2 for 0 mm MgCl
2
and 16 for 10 mm
MgCl
2
. The results are summarized in Table 2. In the
absence of Mg
2+
, the kinetic constants were in agree-
ment with those reported in the literature [23], with
8-oxoG:C being a better substrate because of its lower
K
M
value. The effect of Mg
2+
on K
M
was not high
($ 1.5-fold); however, in the presence of Mg
2+
, K
M
improved for 8-oxoG:C and worsened for 8-oxoG:A.
By contrast, Mg
2+
reduced k
cat

in both cases, possibly
due to the induction of conformational changes in the
DNA molecule that interfere with those required for
catalysis by Fpg [29]. Thus, K
M
values better reflected
the changes in Fpg opposite-base specificity induced by
Mg
2+
in single time point factorial design experiments.
OGG1 generally does not display Michaelis–Menten
kinetics due to the slow release of the reaction product
[30]. However, it is possible to describe the action of
this enzyme by a three-step kinetic scheme (Scheme 1)
and determine two individual rate constants, k
2
and
k
3
, which describe the processes of base excision and
product release, respectively, using single turnover
kinetics for k
2
and burst rate kinetics for k
3
[30,31].
E þ S 
k
1
k

À1
ES !
k
2
EP !
k
3
E þ P Scheme 1
To independently evaluate the effects of ionic
strength and Mg
2+
on the activity and specificity of
OGG1, we measured the apparent values of k
2
and k
3
under conditions of low salt (KP
i
only) and no Mg
2+
,
low salt and 20 mm Mg
2+
, and high salt
(KP
i
+ 150 mm KCl) and no Mg
2+
. These conditions
were selected to represent regions of the factorial

design experiments markedly different in OGG1 speci-
ficity (Fig. 1F), i.e. low preference for 8-oxoG:C versus
8-oxoG:A in low salt and no Mg
2+
(C ⁄ A specificity of
1.5 for DNA glycosylase reaction and 0.78 for AP
lyase reaction) and the increase in the preference for
8-oxoG:C with increasing salt (C ⁄ A specificity of 8.6
for DNA glycosylase reaction and 1.7 for AP lyase
reaction in 150 mm KCl, 0 mm MgCl
2
)orMg
2+
(C ⁄ A
specificity of 18 for DNA glycosylase reaction and 6.7
for AP lyase reaction in 0 mm KCl, 20 mm MgCl
2
).
The results are summarized in Table 3. The k
2
and k
3
constants did not show much variation for 8-oxoG:C
over the set of conditions tested, with a maximum
2.5-fold difference in k
2
and a 2.2-fold difference in k
3
,
and both rate constants improved on addition of

MgCl
2
or KCl. However, an increase in the ionic
strength of Mg
2+
had a pronounced deleterious effect
on k
2
and k
3
for 8-oxoG:A substrates: 20 mm Mg
2+
decreased k
2
by 42-fold and k
3
by 5.5-fold, whereas
150 mm KCl decreased k
2
by 3.9-fold and k
3
by 6.9-
fold. Therefore, physiological concentrations of ionic
strength and divalent cations enhance both base exci-
sion and the turnover of OGG1 cleaving its proper
substrate, 8-oxoG:C, and prevent cleavage of the
improper 8-oxoG:A substrate.
A well-recognized mechanism by which ionic strength
and divalent cations could modulate the activity of
DNA-dependent enzymes is changes in the affinity of

the enzymes for their DNA substrates. For example,
binding of Fpg to damaged DNA shows a bell-shaped
dependence with a peak at $ 100 mm KCl and an
approximately twofold decrease in binding at 0 and
500 mm KCl [32]. Thus, to address the influence of the
reaction conditions on the binding of Fpg and OGG1
to damaged DNA, we determined K
d
values for binding
under the same conditions as used for the kinetic experi-
ments. In these experiments, fluorescence titration was
the method of choice because it allows full control over
the composition of the reaction mixture. To minimize
the impact of protein binding to non-damaged DNA,
shortened 12-mer ligands were used, identical in
sequence to the central part of the 23-mers used in
the kinetic experiments but containing an uncleavable
Table 2. Kinetic constants of cleavage of 8-oxoG:C and 8-oxoG:A
substrates by Fpg in the presence and in the absence of Mg
2+
.
Mean ± SD of three independent experiments.
Substrate
MgCl
2
(mM) K
M
(nM) k
cat
(min

)1
)
k
cat
⁄ K
M
(nM
)1
Æmin
)1
)
8-oxoG:C 0 26 ± 6 6.1 ± 1.2 0.24
10 17 ± 2 1.2 ± 0.2 0.068
8-oxoG:A 0 490 ± 150 3.3 ± 0.7 0.0066
10 700 ± 340 1.0 ± 0.4 0.0015
Factors affecting the specificity of Fpg and OGG1 V. S. Sidorenko et al.
3750 FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS
tetrahydrofuran (THF) moiety instead of 8-oxoG. THF
is a good ligand for Fpg and OGG1, with their affinity
for THF-containing DNA closely paralleling the affinity
for 8-oxoG-containing DNA [23,26], and these particu-
lar ligands have been successfully used to analyze
stopped-flow kinetics for both enzymes [19,20]. The
results of the fluorescence titration experiments are
summarized in Fig. 2. In the absence of MgCl
2
, the
affinity of Fpg for the THF:C ligand was 1.6-fold higher
than for the THF:A ligand. The presence of Mg
2+

had
little effect on the binding of Fpg to the THF:C ligand
and slightly improved binding to the THF:A ligand,
making it comparable with binding to THF:C (Fig. 2B,
groups 1 and 2). Therefore, it is unlikely that the
observed decrease in enzyme activity on 8-oxoG:A and
the concomitant increase in C ⁄ A specificity are due to
differences in binding. In the case of OGG1, the affinity
of the enzyme for THF:C was 3.5-fold higher than for
THF:A in the absence of MgCl
2
and at low ionic
strength. Addition of 150 mm KCl did not change the
situation much, whereas addition of 20 mm MgCl
2
increased the K
d
values for both ligands, with THF:C
affected more than THF:A but still preferred by
OGG1 (Fig. 2B, groups 3–5). As with Fpg, these obser-
vations do not support the idea that binding of the
glycosylase to damaged DNA contributes significantly
to ionic strength and the effects of Mg
2+
on enzyme
specificity.
Table 3. Rate constants of cleavage of 8-oxoG:C and 8-oxoG:A substrates by OGG1 under different conditions. Mean ± SD of 3–5 indepen-
dent experiments.
Conditions
8-oxoG:C 8-oxoG:A

k
2
(min
)1
) k
3
(min
)1
) k
2
(min
)1
) k
3
(min
)1
)
0m
M KCl
0m
M MgCl
2
0mM KGlu
0.50 ± 0.09 0.21 ± 0.11 0.97 ± 0.49 0.011 ± 0.008
0m
M KCl
20 m
M MgCl
2
0mM KGlu

1.1 ± 0.4 0.22 ± 0.03 0.023 ± 0.008 0.0020 ± 0.0013
150 m
M KCl
0m
M MgCl
2
0mM KGlu
1.3 ± 0.6 0.47 ± 0.06 0.25 ± 0.10 0.0016 ± 0.0004
0m
M KCl
0m
M MgCl
2
200 mM KGlu
0.58 ± 0.16 0.14 ± 0.02 1.66 ± 0.68 0.0007 ± 0.001
a
a
Large error is due to fitting to a very shallow-slope linear curve.
[ligand], µM
0
1 2
3
4
5
6
7
Fluorescence, a.u.
0.0
0.5
1.0

1.5
2.0
CA CA CA CA CA CA
Kd, µM
0
2
4
6
8
1 2
3 4 5
6
A
B
Fig. 2. Binding of Fpg and OGG1 to uncleavable THF:C and THF:A ligands under different conditions. (A) A representative experiment show-
ing fluorescence titration of Fpg with a THF:C ligand in the presence of 0 m
M (black circles) or 10 mM (white circles) MgCl
2
. AU, arbitrary
units. (B) Dissociation constants for binding of Fpg (1, 2) and OGG1 (3–6) to THF:C and THF:A ligands (denoted C and A, and represented by
white and black circles, respectively) determined from the fluorescence titration data. The variable components of the buffers included:
25 m
M KP
i
and 50 mM KCl (1), 25 mM KP
i
,50mM KCl and 10 mM MgCl
2
(2), 25 mM KP
i

(3), 25 mM KP
i
and 20 mM MgCl
2
(4), 25 mM KP
i
and 150 mM KCl (5), 25 mM KP
i
and 200 mM KGlu (6) (see also Tables 2 and 3). The mean ± SD of two independent experiments is shown.
V. S. Sidorenko et al. Factors affecting the specificity of Fpg and OGG1
FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3751
Effects of organic anions on the specificity of Fpg
and OGG1
It has been reported previously that the activity and
specificity of some enzymes can depend on the pres-
ence of organic anions in the reaction. For example, a
typical organic anion, glutamate, has been found to
improve the efficiency of DNA synthesis by DNA
polymerase I or its Klenow fragment, as well as their
ability to bypass DNA lesions, in comparison with Cl
)
[33]. Because organic anions represent a major fraction
of total ions and buffering species in the cell, we inves-
tigated how the presence of glutamate affects the activ-
ity and specificity of Fpg and OGG1. Two factorial
design experiments were performed, one with potas-
sium glutamate (KGlu) replacing KCl as a salt in the
presence of KP
i
as the main buffering agent, another

with KGlu as the sole salt and buffer; the Mg
2+
con-
centration was varied in the same way as in the KP
i
–
KCl experiments described above (Table 1, Series 2).
For Fpg, the substitution of KGlu for KCl did not
change the overall dependence of the enzyme activity if
KP
i
was present (Fig. 3A–C). The only notable differ-
ence was a higher activity towards 8-oxoG:C at high
Mg
2+
and salt concentrations compared with when
Cl
)
was the major anion (cf. Figs 1A and 3A). As a
consequence, the specificity of Fpg for 8-oxoG:C versus
8-oxoG:A was highest at 150–200 mm KGlu and 5–
20 mm MgCl
2
, conditions that may better resemble the
cellular environment. If KP
i
was absent (Fig. 4A–C),
Fpg had very low activity at 0 mm KGlu and 0 mm
MgCl
2

, possibly because no ionic strength was pro-
vided (except 1.25 mm KP
i
from the enzyme dilution
buffer). However, KGlu as a sole buffer supported a
substantially high activity of Fpg towards 8-oxoG:C at
all other concentrations of KGlu and MgCl
2
, and
towards 8-oxoG:A at 0–5 mm MgCl
2
. Overall, the
C ⁄ A specificity in this case also increased with increas-
ing salt and MgCl
2
.
In the case of OGG1, replacing KCl with KGlu did
not have much influence on enzyme glycosylase activ-
ity towards 8-oxoG:C (cf. Figs 1D and 3D). The gly-
cosylase activity of OGG1 towards 8-oxoG:A was
highest at 0 mm KGlu and 0 mm MgCl
2
and decreased
at higher concentrations, but, unlike the situation
observed with KCl, it remained essentially unchanged
as the salt concentrations increased (Fig. 3E). As a
result, the C ⁄ A specificity of this reaction was highest
at low to medium concentrations of KGlu (0–50 mm)
and MgCl
2

(0–10 mm), whereas the activity towards
8-oxoG:C was higher. The AP lyase reaction with
8-oxoG:A was efficient only at low Mg
2+
and no
KGlu, whereas with 8-oxoG:C it was in general
agreement with the salt dependence of the glycosylase
reaction; the C ⁄ A specificity was highest at low to
medium KGlu and medium MgCl
2
. In the absence of
KP
i
, the DNA glycosylase and AP lyase activity of
OGG1 towards 8-oxoG:C resembled its activity in the
presence of KP
i
(Fig. 4G,H). However, exclusion of
KP
i
significantly influenced both activities of OGG1
with 8-oxoG:A; a more or less efficient glycosylase
reaction was observed only at 0 mm KGlu and 0–
15 mm MgCl
2
, whereas the AP lyase reaction required
0–100 mm KGlu and 0–5 mm MgCl
2
. The C ⁄ A speci-
ficity of the glycosylase reaction in the absence of KP

i
was usually higher than in the presence of KP
i
due to
less efficient cleavage of 8-oxoG:A; the highest specific-
ity was observed at 0 mm KGlu + 20 mm MgCl
2
and
200 mm KGlu + 0 mm MgCl
2
, where cleavage of
8-oxoG:A was minimal. The overall C ⁄ A specificity of
the AP lyase reaction was highest at high KGlu and
low to intermediate MgCl
2
concentrations. Interest-
ingly, OGG1, unlike Fpg, displayed robust activity on
both substrates in the absence of KP
i
, KGlu and
MgCl
2
.
To dissect the kinetic contribution of a high KGlu
concentration to the specificity of OGG1, we also deter-
mined the values k
2
and k
3
with both 8-oxoG:C and

8-oxoG:A in the presence of 25 mm KP
i
and 200 mm
KGlu (C ⁄ A specificity 9.0 for the glycosylase reaction,
15 for the AP lyase reaction). As shown in Table 3, in
comparison with KP
i
only, the addition of KGlu had a
minimal effect on either rate constant in the case of
8-oxoG:C (a 1.2-fold increase in k
2
and an 1.5-fold
decrease in k
3
), and even improved the k
2
value for
8-oxoG:A by 1.7-fold. However, this was accompanied
by a 16-fold decrease in k
3
, indicating that the enzyme
turnover on 8-oxoG:A slows significantly, contributing
to a decrease in the efficiency of its cleavage by OGG1.
Moreover, fluorescence titration analysis of OGG1
binding to uncleavable THF:C and THF:A damaged
ligands showed that although KGlu did not affect the
affinity of the enzyme for the THF:C ligand, its affinity
for the THF:A ligand decreased at least 2.3-fold in com-
parison with the reactions (C⁄ A specificity for binding
was 3.5 for 25 mm KP

i
, 2.8 for 25 mm KP
i
+ 150 mm
KCl, and 8.6 for 25 mm KP
i
+ 200 mm KGlu). Thus,
the presence of KGlu may also disfavor the 8-oxoG:A
substrate at the level of binding.
Polyamines, crowding agents and some purine
analogs do not affect the activity and specificity
of 8-oxoguanine-DNA glycosylases
Several factors that may, in principle, affect the effi-
ciency of 8-oxoG excision by Fpg and OGG1 have
Factors affecting the specificity of Fpg and OGG1 V. S. Sidorenko et al.
3752 FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS
never been investigated. Nucleic acids in bacteria and
human cells are bound to polyamines (spermine, sper-
midine and putrescine), abundant products of amino
acid metabolism with important structural and regula-
tory functions [34]. Because polyamine binding affects
the structure of nucleic acids and the availability of
their hydrogen-bond donors and acceptors, the activi-
ties of DNA-dependent enzymes may be influenced by
polyamine binding to their DNA substrates; for exam-
ple, polyamines activate poly(ADP-ribose) polymerase
[35] and improve the fidelity of HIV-1 reverse trans-
criptase [36]. We investigated the cleavage of 8-oxoG:C
and 8-oxoG:A substrates by Fpg (not shown) and
OGG1 (Fig. 5A,B) in the presence of 0–1000 lm sper-

mine or spermidine (Table 1, Series 3) and varying
concentrations of MgCl
2
. In the absence of Mg
2+
,
spermine slightly ($ 1.5-fold) increased the specificity
of Fpg due to a corresponding decrease in activity on
8-oxoG:A. No significant influence of polyamines on
OGG1 activity was observed, except that AP lyase
activity on 8-oxoG:C was approximately twofold
higher in 1 mm spermine (but not spermidine), possibly
due to the chemical degradation of AP sites by
polyamines [37]. Overall, polyamines had minimal
0
10
20
30
40
50
0
50
100
150
200
0
5
10
15
20

[
P
]
,
n
M
KG
l
u
,
m
M
M
g
C
l
2
,
m
M
0
5
10
15
0
50
100
150
200
0

5
10
15
20
[
P
]
,
n
M
K
G
l
u
,
m
M
M
g
C
l
2
,
m
M
0
10
20
30
40

0
50
100
150
200
0
5
10
15
20
C
/
A
K
G
l
u
,
m
M
M
g
C
l
2
,
m
M
0
10

20
30
40
50
0
50
100
150
200
0
5
10
15
20
[
P
]
,
n
M
KG
l
u
,
m
M
M
g
C
l

2
,
m
M
0
2
4
6
8
10
12
0
50
100
150
200
0
5
10
15
20
[
P
]
,
n
M
K
G
l

u
,
m
M
M
g
C
l
2
,
m
M
0
2
4
6
8
10
0
50
100
150
200
0
5
10
15
20
C
/

A
K
G
l
u
,
m
M
M
g
C
l
2
,
m
M
0
5
10
15
20
25
30
0
50
100
150
200
0
5

10
15
20
[
P
]
,
n
M
K
G
l
u
,
m
M
M
g
C
l
2
,
m
M
0
2
4
6
8
0

50
100
150
200
0
5
10
15
20
[
P
]
,
n
M
KG
l
u
,
m
M
M
g
C
l
2
,
m
M
0

5
10
15
0
50
100
150
200
0
5
10
15
20
C
/
A
K
G
l
u
,
m
M
M
g
C
l
2
,
m

M
ABC
D
EF
GHI
Fig. 3. Activity and specificity of Fpg and OGG1 in buffers with different concentrations of KGlu and MgCl
2
in the presence of KP
i
. (A–C)
Fpg, (D–F) glycosylase activity of OGG1, (G–I) AP lyase activity of OGG1. The extent of cleavage of 8-oxoG:C (A, D, G) or 8-oxoG:A (B, E, H)
or the C ⁄ A specificity (C, F, I) is plotted against the concentrations of the salts. [P], product concentration. Note the different scales in panels
(A, D, G) compared with (B, E, F). The means of two independent experiments are shown.
V. S. Sidorenko et al. Factors affecting the specificity of Fpg and OGG1
FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3753
influence on the activity and specificity of both Fpg
and OGG1.
Another factor that can seriously influence the activ-
ities of various enzymes in the cell is its crowding with
macromolecular agents [38], and crowding has been
shown to modulate DNA-dependent enzymes such as
DNA ligases [39] or restriction endonucleases [40].
Poly(ethylene glycol) fractions of differing average
molecular masses are widely used as crowding agents
in enzyme kinetics. We investigated the activity of Fpg
and OGG1 towards 8-oxoG:C and 8-oxoG:A in the
presence of 0–5% poly(ethylene glycol) 4000 or 8000
and varying concentrations of MgCl
2
(Table 1, Series 4)

and found only marginal differences for any of the
enzyme–substrate pairs [Fig. 5C shows an example of
DNA glycosylase activity of OGG1 with the range
of poly(ethylene glycol) 8000 and 0 mm MgCl
2
].
Therefore, macromolecular crowding is likely to be of
little importance for the function of these two
enzymes.
In addition, we analyzed the effect of two low
molecular mass compounds, biotin and caffeine, on
the activity of Fpg and OGG1. Biotin can be regarded
as a structural mimic of 8-oxopurines [41], and avidin,
0
10
20
30
40
50
60
0
50
100
150
200
0
5
10
15
20

[
P
]
,
n
M
KG
l
u
,
m
M
M
g
C
l
2
,
m
M
0
5
10
15
20
25
30
0
50
100

150
200
0
5
10
15
20
[
P
]
,
n
M
K
G
l
u
,
m
M
M
g
C
l
2
,
m
M
0
10

20
30
40
50
0
50
100
150
200
0
5
10
15
20
C
/
A
K
G
l
u
,
m
M
M
g
C
l
2
,

m
M
0
10
20
30
40
50
0
50
100
150
200
0
5
10
15
20
[
P
]
,
n
M
K
G
l
u
,
m

M
M
g
C
l
2
,
m
M
0
5
10
15
20
0
50
100
150
200
0
5
10
15
20
[
P
]
,
n
M

K
G
l
u
,
m
M
M
g
C
l
2
,
m
M
0
5
10
15
20
25
30
35
0
50
100
150
200
0
5

10
15
20
C
/
A
K
G
l
u
,
m
M
M
g
C
l
2
,
m
M
0
10
20
30
40
0
50
100
150

200
0
5
10
15
20
[
P
]
,
n
M
K
G
l
u
,
m
M
M
g
C
l
2
,
m
M
0
2
4

6
8
10
12
14
0
50
100
150
200
0
5
10
15
20
[
P
]
,
n
M
KG
l
u
,
m
M
M
g
C

l
2
,
m
M
0
10
20
30
40
50
60
0
50
100
150
200
0
5
10
15
20
C
/
A
K
G
l
u
,

m
M
M
g
C
l
2
,
m
M
ABC
DEF
GHI
Fig. 4. Activity and specificity of Fpg and OGG1 in buffers with different concentrations of KGlu and MgCl
2
in the absence of KP
i
. (A–C) Fpg,
(D–F) glycosylase activity of OGG1, (G–I) AP lyase activity of OGG1. The extent of cleavage of 8-oxoG:C (A, D, G) or 8-oxoG:A (B, E, H) or the
C ⁄ A specificity (C, F, I) is plotted against the concentrations of the salts. [P], product concentration. Note the different scales in (A, D, G)
compared with (B, E, F). Means of two independent experiments are shown.
Factors affecting the specificity of Fpg and OGG1 V. S. Sidorenko et al.
3754 FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS
a well-known biotin-binding protein, has been shown
by X-ray crystallography to bind 8-oxopurines in its
biotin-binding site [42]; thus, the possibility of biotin
association with 8-oxoG-binding sites of DNA glycosy-
lases could not be excluded. Caffeine, the most widely
ingested xenobiotic purine base in the world, appar-
ently influences several pathways of DNA repair

through mechanisms that are not fully understood
[43]. We analyzed the ability of Fpg and OGG1 to
cleave their substrates in the presence of up to 20 mm
biotin or caffeine. However, no effect was found
except for a slight ($ 30%) inhibition of Fpg at the
highest caffeine concentration used (data not shown).
Therefore, biotin and caffeine are unlikely to influence
the activities of these enzymes in vivo.
Discussion
The substrate specificity of DNA glycosylases has been
subject to a number of studies, yet the results are often
conflicting. For example, Fpg has been reported to
excise more than 20 different damaged bases from oli-
gonucleotide substrates [44], whereas the excision from
damaged genomic DNA has been reported only for
8-oxoG, 4,6-diamino-5-formamidopyrimidine, 2,4-dia-
mino-6-oxo-5-formamidopyrimidine and 2,4-diamino-
6-oxo-5N-methyl-5-formamidopyrimidine [45,46]. The
relative activity of Fpg on substrates containing
different bases opposite 8-oxoG also seemingly varies
depending on the assay used [19,23]. It is clear that
when an enzyme can process several substrates with
comparable efficiencies, as is the case for almost all
DNA glycosylases [47], the preferences for each sub-
strate may depend on the reaction conditions to differ-
ent degrees. The influence of the reaction conditions
on various aspects of substrate specificity of DNA
glycosylases has been given little attention, but there
are reasons to believe that the impact of ionic strength
and divalent cations may be significant. In one recent

study, submillimolar concentrations of Mg
2+
have
been shown to stimulate the excision of hypoxanthine
but not of 1,N
6
-ethenoadenine by murine methyl-
purine-DNA glycosylase (MPG) [48]. Regarding the
opposite-base specificity, discrimination of the opposite
base by human endonuclease III is strongly dependent
on Mg
2+
concentrations, approaching its maximum at
10–20 mm MgCl
2
[49].
Our study explicitly addressed the opposite-base
specificity of two 8-oxoguanine-DNA glycosylases, the
only DNA glycosylases for which the preference for a
particular base opposite the lesion has been proved to
play a biologically important role [7–9]. Both Fpg and
OGG1 display a strong preference for 8-oxoG:C in
comparison with 8-oxoG:A [21,23,26,50]. We were
interested in a systematic analysis of this opposite-base
preference, and, in particular, how it may change
under conditions approximating the intracellular envi-
ronment. Two principal factors that affected the oppo-
site-base specificity of Fpg and OGG1 were general
ionic strength and Mg
2+

concentration. In general, the
specificity of both enzymes was highest when these fac-
tors approached physiological values. The reason for
the increase in specificity was a pronounced decrease
in the activity of Fpg and OGG1 on 8-oxoG:A at high
Mg
2+
and ionic strength, whereas most of the activity
on 8-oxoG:C was retained under these conditions.
At least for OGG1, we observed only a modest
decrease in the affinity for both THF:C and THF:A
uncleavable ligands with increasing salt concentration.
In the case of Fpg, it has been reported that binding
0
1
10
100
1000
% Activity
0
50
100
150
200
Spermine, µM
0
1
10
100
1000

PEG 8000, %
0
0.1
1
10
A B C
Fig. 5. Activity of OGG1 in the presence of polyamines and crowding agents. (A) DNA glycosylase and (B) AP lyase activity on the 8-oxoG:C
substrate in the presence of spermine (0 m
M MgCl
2
). (C) DNA glycosylase activity on the 8-oxoG:C substrate in the presence of poly(ethyl-
ene glycol) 8000 (0 m
M MgCl
2
). The activity in the presence of spermine or poly(ethylene glycol) is normalized to the same activity in their
absence (100%); the scale is the same in all panels. Means ± SD of three independent experiments are shown; in some cases, the error
bars are hidden by the symbols.
V. S. Sidorenko et al. Factors affecting the specificity of Fpg and OGG1
FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3755
of this enzyme to uncleavable damaged DNA ligands
is also moderately affected by salt concentration
(approximately twofold difference between the best
and the worst binding in the 0–500 mm KCl range)
[32]. Therefore, general affinity does not seem to con-
tribute much to the effect of ionic strength on glycosy-
lase activity and specificity. However, ionic strength
may possibly have a selective effect on some stages of
multistage lesion recognition by Fpg and OGG1
[19,20]. Although no structural information on Fpg or
OGG1 complexed with DNA containing A opposite

the lesion is available, the structures of both enzymes
complexed with undamaged DNA show that initial
recognition of the lesion involves mostly weak non-
specific interactions partly mediated through a water
layer [51,52]. Such protein–DNA interactions are easily
competed out by small cations [53]. Because the initial
recognition complexes exist for longer during process-
ing of 8-oxoG:A by either Fpg or OGG1, whereas
with 8-oxoG:C the reaction quickly proceeds to its
catalytic steps [19,20], the effect of electrostatic screen-
ing by higher ionic strength may be more pronounced
with 8-oxoG:A substrates; it is also possible that
electrostatic interactions may stabilize catalytically
inactive conformations of the enzyme in the case of
8-oxoG:A.
Unlike 8-oxoG:C pairs that exist in a conventional
anti ⁄ anti conformation [54], the 8-oxoG deoxynucleo-
tide in 8-oxoG:A mispairs prefers a syn conformation
due to steric repulsion between the O
8
atom and the
5¢-phosphate [3,55]. Thermodynamic and modeling
studies suggest that 8-oxoG may exist in a syn⁄ anti equi-
librium when paired with A [56,57]. Mg
2+
is known to
induce conformational transitions in nucleic acids, pos-
sibly by selective stabilization ⁄ destabilization of one of
the conformations; for example, submillimolar concen-
trations of Mg

2+
induce a transition of poly(dG-
m
5
dC)Æpoly(dG-m
5
dC) from the B-DNA to the Z-DNA
form [58]. Thus, the apparent increase in the C ⁄ A speci-
ficity of Fpg and OGG1 in the presence of MgCl
2
may
be caused by a shift in the conformational equilibrium
of the 8-oxoG:A pair towards 8-oxoG(syn):A(anti),
which may be poorly recognized by the enzyme [23]. In
the case of OGG1, another possible mode of Mg
2+
action may be via binding to a metal-binding site formed
at the OGG1 ⁄ DNA interface [16], with a potential to
destabilize the catalytically competent conformation of
the enzyme–substrate complex with an adenine base
opposite the lesion. The effect of magnesium may be
aggravated by the tendency of its solvated ions to form
multiple water bridges with adjacent positions in DNA
[59], possibly influencing conformational dynamics of
damaged DNA during recognition and catalysis by Fpg
or OGG1. Regarding OGG1, our results are in agree-
ment with a recent report [60] that a high Mg
2+
concen-
tration inhibits the AP lyase activity of the enzyme more

than its DNA glycosylase activity.
The nature of anions in the reaction mixture did not
affect the general specificity, although some details were
notably different between reactions performed in the
presence of KCl and KGlu. For example, KGlu sup-
ported the DNA glycosylase activity of OGG1 on
8-oxoG:A over a much wider range of salt and Mg
2+
concentrations than KCl did, and tended to sustain the
activity of Fpg and the AP lyase activity of OGG1 at
low Mg
2+
better than KCl. At the same time, high
KGlu concentrations selectively decreased the turnover
of OGG1 on the 8-oxoG:A substrate and the affinity of
OGG1 for the uncleavable THF:A ligand. Some anions
are known to influence the activity of DNA glycosylases;
for example, phosphate is a competitive inhibitor of Fpg
with K
i
$ 10 mm [61], and we observed that the activity
of Fpg on 8-oxoG:C in the absence of KP
i
was generally
higher than in its presence. However, it seems that the
overall differences between Cl
)
and glutamate are not
decisive for the opposite-base specificity of Fpg and
OGG1. Also, although the buffering capacity of KGlu

at pH 7.5 is much weaker than that of KP
i
, the omission
of KP
i
had a rather minor effect on the specificity of
both enzymes.
Even less important for the activity and opposite-base
specificity of Fpg and OGG1 were polyamines (spermine
and spermidine), crowding agents [poly(ethylene glycol)],
and small heterocyclic molecules (biotin and caffeine).
The effect that polyamines and crowding agents may
have on the activity of DNA-dependent enzymes in the
cell is often under-appreciated, and some enzymes may
be notably activated or inhibited by these factors
[35,36,39,40]. However, this is apparently not the case
with Fpg and OGG1, and the kinetic parameters deter-
mined in the absence of polyamines and crowding
agents need not be corrected when considering the intra-
cellular environment. Biotin and caffeine were selected
for study as molecules that may, in principle, resemble
purine substrates of Fpg and OGG1 and be bound in
the active sites of these enzymes. Modulation of the
activity of DNA glycosylases by small, naturally
encountered heterocyclic molecules is not without
precedent: several DNA glycosylases are inhibited or
activated by their substrate bases in a free form [62–65],
other nucleobases [66] and even by heterocyclic mole-
cules only distantly resembling the target bases (e.g.
MPG is inhibited by pterine) [65]. Again, this was not

observed with Fpg, which is also not inhibited by free
8-oxoG [21,60], and OGG1, which has been reported to
be inhibited by 8-oxoG [60].
Factors affecting the specificity of Fpg and OGG1 V. S. Sidorenko et al.
3756 FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS
The problem of discriminating against the DNA ele-
ments that are close to the substrates but should not be
processed is not unique for DNA glycosylases. Well-
known phenomena are the misinsertion of an incorrect
dNMP by DNA polymerases [67,68] or ‘star activity’ of
restriction endonucleases [69]. In all these cases, the
fidelity of the enzymatic reaction may be significantly
influenced by the presence of various compounds in the
reaction mixture. Generally, lower ionic strength, high
crowding and divalent cations other than Mg
2+
tend to
facilitate non-specific reactions, whereas high ionic
strength and Mg
2+
increase the fidelity. In this respect,
some intracellular factors (ionic composition) favor cor-
rect, physiologically relevant substrates, whereas the
others (crowding) act in the opposite direction. As our
results indicate, inside the cell, the preference of Fpg
and OGG1 for processing substrates with C as
compared with those containing A opposite the lesion,
significantly relies on the high intracellular ionic
strength and Mg
2+

concentrations.
Experimental procedures
Enzymes and oligonucleotides
Fpg and OGG1 were overexpressed in E. coli and purified
as described [17,70]. The concentrations of the active
enzymes were determined by borohydride trapping and
burst-phase kinetics as described previously [19,31]. Phage
T4 polynucleotide kinase was purchased from New England
Biolabs (Beverly, MA, USA). Modified oligonucleotides
d(CCTTCXCTCCTT) (X = THF) and d(CTCTCCCTTC
XCTCCTTTCCTCT) (X = 8-oxoG) and complementary
oligonucleotides placing C or A opposite the lesion were
synthesized from commercially available phosphoramidites
(Glen Research, Sterling, VA, USA) and purified by
reverse-phase HPLC on a PRP-1 column (Hamilton, Reno,
NV, USA). The modified strand was
32
P-labeled using T4
polynucleotide kinase, purified by reverse-phase chromato-
graphy on a NenSorb C
18
sorbent (Applera, Norwalk, CT,
USA) according to the manufacturer’s instructions, and
annealed to a twofold molar excess of the appropriate com-
plementary strand.
Cleavage assay
The standard reaction mixture [10 lL, 20 lL if poly(ethyl-
ene glycol) was present] included 50 nm
32
P-labeled duplex

substrate, 25 mm KP
i
(pH 7.5; omitted in some specified
cases), 1 mm dithiothreitol and Fpg (5 nm) or OGG1
(10 nm for the glycosylase activity assay, 100 nm for the
AP lyase activity assay). The enzymes were diluted to
10-fold working concentrations in 12.5 mm KP
i
(pH 7.5)
supplemented with 0.5 mgÆmL
)1
BSA. The varied compo-
nents of the reaction mixture included KCl, KGlu, MgCl
2
,
spermine or spermidine and poly(ethylene glycol) (4000 or
8000); the components included in complete factorial design
experiments are summarized in Table 1. When KGlu was
used as a component of the reaction mixture, its stock was
first brought to pH 7.5 by titration with glutamic acid and
then used to prepare the reaction mixtures; the concen-
trations are reported with respect to glutamate anion. If
necessary, the reaction mixture was supplemented with
0.1–20 mm biotin or caffeine. The reaction was initiated by
adding the enzyme (1 ⁄ 10 volume of the reaction mixture)
and allowed to proceed for 5 min (Fpg) or 10 min (OGG1).
In the case of Fpg, the reaction was terminated by mixing
with 1 ⁄ 2 vol. of formamide loading dye [71] and heating
for 5 min at 95 °C. To measure the glycosylase activity of
OGG1, the reaction was terminated by heating for 30 min

at 95 °C (AP sites are fully cleaved under these conditions)
[72], mixed with 1 ⁄ 2 vol. of formamide loading dye and
heated again for 2 min at 95 °C. To measure the AP lyase
activity of OGG1, the reaction was terminated by mixing
with 1 ⁄ 2 vol. of formamide loading dye followed by heating
for 1 min at 95 °C and storage on ice until analysis. The
reaction products were separated by denaturing PAGE (20%
polyacrylamide, 7.2 m urea) and quantified using a Molecu-
lar Imager FX system (Bio-Rad, Hercules, CA, USA).
Kinetic constant determination
The reaction mixtures were as in the standard assay, except
that the components specified in the Results were added, and
the concentrations of the substrate and enzyme varied as
required for the respective kinetics. To calculate the steady-
state catalytic constants (K
M
and k
cat
) for Fpg, the enzyme
was used at 1 nm (8-oxoG:C) or 10 nm (8-oxoG:A) and the
substrate concentrations varied from 1.5 to 100 nm
(8-oxoG:C) or from 40 to 2700 nm (8-oxoG:A). To deter-
mine the base excision rate constant (k
2
) for OGG1 in the
single turnover assay, the reaction mixture included 10 nm
substrate and 100 or 2000 nm enzyme (for 8-oxoG:C and
8-oxoG:A, respectively). The reaction mixture was incubated
at 8 °C for 0.5–30 min (8-oxoG:C) or 1–90 min (8-oxoG:A)
and the glycosylase activity was assayed as above. To deter-

mine the product release rate constant (k
3
) for OGG1 in the
burst rate assay, the reaction mixture included 50 nm
substrate and 10 nm enzyme. The reaction mixture was
incubated at 37 °C for 0.5–5 min and the glycosylase activity
was assayed. All reported kinetic constants were obtained by
non-linear least-square fitting using sigmaplot v. 8.0 soft-
ware (Systat Software, San Jose, CA, USA).
Fluorimetric titration
Binding of Fpg and OGG1 to the THF-containing ligands
was assayed by following tryptophan fluorescence of the
enzymes using an SFM-25 spectrofluorimeter (Kontron,
V. S. Sidorenko et al. Factors affecting the specificity of Fpg and OGG1
FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3757
Munich, Germany), equipped with a 80 lL cuvette thermo-
stated at 15 °C. The fluorescence was excited at k
ex
=
290 nm, and the emission was recorded at k
em
= 300–
360 nm. The reaction mixtures were the same as described
under ‘Kinetic constants determination’ except the enzyme
concentrations were different (1 lm Fpg, 3.6 lm OGG1)
and the oligonucleotide ligand was added in small incre-
ments until the binding curve was saturated. K
d
values were
extracted by fitting the fluorescence data to a single-site

ligand-binding model [70].
Acknowledgments
This work was supported in part by grants from the
Presidium of the Russian Academy of Sciences (Pro-
gram 10.1), Russian Foundation for Basic Research
(07-04-00395, 08-04-00596), Russian Ministry of
Science and Education (02.512.11.2194), and an Inte-
grative Grant from the Siberian Branch of the Russian
Academy of Sciences (Program 6). We thank Dr
N. A. Kuznetsov (Institute of Chemical Biology and
Fundamental Medicine) for help with collecting the
fluorescence data.
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