We investigated the influence of various factors, including ionic strength, the presence of Mg2+ and organic anions, poly-amides, crowding agents and two small heterocyclic compounds biot
Trang 1Escherichia coli and human 8-oxoguanine-DNA
glycosylases
Viktoriya S Sidorenko1, Grigory V Mechetin1, Georgy A Nevinsky1,2and Dmitry O Zharkov1,2
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 · 106 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: dzharkov@niboch.nsc.ru
(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 Mg2+ 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 Mg2+ concentration, whereas the activity on 8-oxoG:C was much more stable, resulting in higher oppo-site-base specificity when salt and Mg2+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 Mg2+
Abbreviations
8-oxoG, 8-oxo-7,8-dihydroguanine; AP, apurinic ⁄ apyrimidinic; KGlu, potassium glutamate; THF, tetrahydrofuran.
Trang 2repair 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:Cfi 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 Mg2+, the nature of the
buffer-ing agents, the presence of competbuffer-ing 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, Mg2+ 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 5 mm, the major intracellular monovalent cation is K+ ( 200–250 mm), free diva-lent cations are mostly Mg2+ ( 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 MgCl2, whereas the buffering agent (potassium phosphate,
KPi) 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
3 Spermine or spermidine 0, 1, 10, 100, 1000 l M
4 Poly(ethylene glycol)
4000 or 8000
0, 0.05, 0.1, 0.2, 0.5,
1, 2, 5%
Trang 3the 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 Mg2+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 MgCl2
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 MgCl2 Interestingly, the AP lyase activity of OGG1 on 8-oxoG:A at any given
0
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Fig 1 Activity and specificity of Fpg and OGG1 in buffers with different concentrations of KCl and MgCl2 (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.
Trang 4concentration 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
MgCl2 and 50–150 mm KCl (Fig 1C), for OGG1
glycosylase activity, at 5–10 mm MgCl2 and 150–
200 mm KCl (Fig 1F) and for its AP lyase activity, at
10–20 mm MgCl2and 100–200 mm KCl (Fig 1I)
To analyze the impact of Mg2+ 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 abspres-ence of 10 mm MgCl2 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 MgCl2 and 16 for 10 mm
MgCl2 The results are summarized in Table 2 In the
absence of Mg2+, 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
KM value The effect of Mg2+ on KM was not high
( 1.5-fold); however, in the presence of Mg2+, KM
improved for 8-oxoG:C and worsened for 8-oxoG:A
By contrast, Mg2+reduced kcatin 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, KM values better reflected
the changes in Fpg opposite-base specificity induced by
Mg2+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, k2 and
k3, which describe the processes of base excision and
product release, respectively, using single turnover
kinetics for k2and burst rate kinetics for k3[30,31]
Eþ S k1
k 1
ES!k2
EP!k3
To independently evaluate the effects of ionic strength and Mg2+ on the activity and specificity of OGG1, we measured the apparent values of k2 and k3 under conditions of low salt (KPionly) and no Mg2+, low salt and 20 mm Mg2+, and high salt (KPi+ 150 mm KCl) and no Mg2+ 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 Mg2+(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 MgCl2) or Mg2+(C⁄ A specificity of 18 for DNA glycosylase reaction and 6.7 for AP lyase reaction in 0 mm KCl, 20 mm MgCl2) The results are summarized in Table 3 The k2 and k3 constants did not show much variation for 8-oxoG:C over the set of conditions tested, with a maximum 2.5-fold difference in k2and a 2.2-fold difference in k3, and both rate constants improved on addition of MgCl2 or KCl However, an increase in the ionic strength of Mg2+ had a pronounced deleterious effect
on k2 and k3 for 8-oxoG:A substrates: 20 mm Mg2+ decreased k2 by 42-fold and k3 by 5.5-fold, whereas
150 mm KCl decreased k2 by 3.9-fold and k3 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 Kdvalues 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
(m M) KM(n M) kcat(min)1)
k cat ⁄ K M
(n M )1Æmin)1)
Trang 5tetrahydrofuran (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 MgCl2, the
affinity of Fpg for the THF:C ligand was 1.6-fold higher
than for the THF:A ligand The presence of Mg2+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 MgCl2 and at low ionic strength Addition of 150 mm KCl did not change the situation much, whereas addition of 20 mm MgCl2 increased the Kd 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 Mg2+ 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
0 m M KCl
0 m M MgCl2
0 m M KGlu
0 m M KCl
20 m M MgCl2
0 m M KGlu
150 m M KCl
0 m M MgCl2
0 m M KGlu
0 m M KCl
0 m M MgCl2
200 m M KGlu
a Large error is due to fitting to a very shallow-slope linear curve.
0 1 2 3 4 5 6 7
0.0
0.5
1.0
1.5
2.0
0
2
4
6
8
1 2 3 4 5 6
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 m M (white circles) MgCl2 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 m M KCl (1), 25 m M KP i , 50 m M KCl and 10 m M MgCl 2 (2), 25 m M KP i (3), 25 m M KP i and 20 m M MgCl 2 (4), 25 m M KP i
and 150 m M KCl (5), 25 m M KPiand 200 m M KGlu (6) (see also Tables 2 and 3) The mean ± SD of two independent experiments is shown.
Trang 6Effects 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 specificactiv-ity 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 KPi as the main buffering agent, another
with KGlu as the sole salt and buffer; the Mg2+
con-centration was varied in the same way as in the KPi–
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
KPi was present (Fig 3A–C) The only notable
differ-ence was a higher activity towards 8-oxoG:C at high
Mg2+ 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 MgCl2, conditions that may better resemble the
cellular environment If KPi was absent (Fig 4A–C),
Fpg had very low activity at 0 mm KGlu and 0 mm
MgCl2, possibly because no ionic strength was
pro-vided (except 1.25 mm KPi 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 MgCl2, and
towards 8-oxoG:A at 0–5 mm MgCl2 Overall, the
C⁄ A specificity in this case also increased with
increas-ing salt and MgCl2
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 MgCl2 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 MgCl2 (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 Mg2+ 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 MgCl2 In the absence of
KPi, the DNA glycosylase and AP lyase activity of OGG1 towards 8-oxoG:C resembled its activity in the presence of KPi (Fig 4G,H) However, exclusion of
KPi 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 MgCl2, whereas the AP lyase reaction required 0–100 mm KGlu and 0–5 mm MgCl2 The C⁄ A speci-ficity of the glycosylase reaction in the absence of KPi was usually higher than in the presence of KPi due to less efficient cleavage of 8-oxoG:A; the highest specific-ity was observed at 0 mm KGlu + 20 mm MgCl2 and
200 mm KGlu + 0 mm MgCl2, 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 MgCl2 concentrations Interest-ingly, OGG1, unlike Fpg, displayed robust activity on both substrates in the absence of KPi, KGlu and MgCl2
To dissect the kinetic contribution of a high KGlu concentration to the specificity of OGG1, we also deter-mined the values k2 and k3 with both 8-oxoG:C and 8-oxoG:A in the presence of 25 mm KPi 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 KPionly, 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 k2 and an 1.5-fold decrease in k3), and even improved the k2 value for 8-oxoG:A by 1.7-fold However, this was accompanied
by a 16-fold decrease in k3, 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 KPi, 2.8 for 25 mm KPi+ 150 mm KCl, and 8.6 for 25 mm KPi+ 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
Trang 7never 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 MgCl2 In the absence of Mg2+, 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
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KGlu,
mM
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2 , mM
0 5 10 15
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0 5 10 15 20
KGlu , mM
MgCl
2 , mM
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.
Trang 8influence 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 MgCl2(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 MgCl2] 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,
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MgCl
2 , mM
0
10
20
30
40
50
0 50 100 150 200
0
5
10
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20
KGlu , mM
MgCl
2 , mM
0 5 10 15 20
0 50 100 150 200
0 5 10 15 20
MgCl
2 , mM
0 5 10 15 20 25 30 35
0 50 100 150 200
0 5 10 15 20
KGlu,
mM
MgCl
2 , mM
0
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20
30
40
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0
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KGlu , mM
MgCl
2 , mM
0 2 4 6 8 10 12 14
0 50 100 150 200
0 5 10 15 20
KGlu,
mM
MgCl
2 , mM
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0 50 100 150 200
0 5 10 15 20
KGlu , mM
MgCl
2 , mM
Fig 4 Activity and specificity of Fpg and OGG1 in buffers with different concentrations of KGlu and MgCl2in the absence of KPi (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.
Trang 9a 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 Mg2+ have been shown to stimulate the excision of hypoxanthine but not of 1,N6-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 Mg2+concentrations, approaching its maximum at 10–20 mm MgCl2[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 Mg2+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
Mg2+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
0
50
100
150
200
Spermine, µ M
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 MgCl2) (C) DNA glycosylase activity on the 8-oxoG:C substrate in the presence of poly(ethyl-ene glycol) 8000 (0 m M MgCl2) 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.
Trang 10of 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 O8 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] Mg2+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 Mg2+ induce a transition of
poly(dG-m5dC)Æpoly(dG-m5dC) 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 MgCl2may
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 Mg2+
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 Mg2+ 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 Mg2+ concentrations than KCl did, and tended to sustain the activity of Fpg and the AP lyase activity of OGG1 at low Mg2+ 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 Ki 10 mm [61], and we observed that the activity
of Fpg on 8-oxoG:C in the absence of KPiwas 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 KPi, the omission
of KPi 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]