(Received for publication, March 20, 1995; and in revised form, June 7, 1995)
From the
DNA glycosylases catalyze scission of the N-glycosylic bond linking a damaged base to the DNA sugar phosphate backbone. Some of these enzymes carry out a concomitant abasic (apyrimidinic/apurinic (AP)) lyase reaction at a rate approximately equal to that of the glycosylase step. As a generalization of the mechanism described for T4 endonuclease V, a repair glycosylase/AP lyase that is specific for ultraviolet light-induced cis-syn pyrimidine dimers, a hypothesis concerning the mechanism of these repair glycosylases has been proposed. This hypothesis describes the initial action of all DNA glycosylases as a nucleophilic attack at the sugar C-1` of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. It is proposed that the enzymes that are only glycosylases differ in the chemical nature of the attacking nucleophile from the glycosylase/AP lyases. Those DNA glycosylases, which carry out the AP lyase reaction at a rate approximately equal to the glycosylase step, are proposed to use an amino group as the nucleophile, resulting in an imino enzyme-DNA intermediate. The simple glycosylases, lacking the concomitant AP lyase activity, are proposed to use some nucleophile from the medium, e.g. an activated water molecule. This paper reports experimental tests of this hypothesis using five representative enzymes, and these data are consistent with this hypothesis.
DNA glycosylases initiate the cellular DNA base excision repair
pathway. In this pathway, damaged or inappropriate bases are removed
from the DNA as free bases by cleavage of the N-glycosylic
bond (reviewed in (1) and (2) ). These enzymes have
been classified into two groups based on the types of products they
generate (Fig. 1). The simple glycosylases are limited to the N-glycosylic bond cleavage, the product of which is an abasic
site (Fig. 1B). The glycosylase/AP ()lyases
carry out the glycosylase reaction and also catalyze a subsequent AP
lyase activity at a rate approximately equal to the glycosylase rate (Fig. 1, C-E). T4 endonuclease V, Micrococcus
luteus UV endonuclease, Escherichia coli endonuclease
III, and E. coli formamidopyrimidine (Fapy) glycosylase (FPG)
are members of the first
group(3, 4, 5, 6, 7, 8, 9, 10) .
Uracil DNA glycosylase (UDG) and adenine DNA glycosylase (MutY) from E. coli, mammalian N-methyl purine glycosylase (MPG),
and 3-methyladenine DNA glycosylase of E. coli are examples of
simple glycosylases (11, 12, 13, 14) . To date the
underlying mechanistic differences between these two groups of enzymes
have not been described.
Figure 1: Schematic representation of the overall reaction pathway for DNA glycosylases and glycosylase/AP lyases.
T4 endonuclease V, a repair
glycosylase/lyase specific for ultraviolet light-induced cis-syn cyclobutane pyrimidine dimers, was the first DNA glycosylase to
have its structure determined by x-ray crystallography and its active
site residues
identified(15, 16, 17, 18, 19, 20, 21, 22) .
T4 endonuclease V uses the N-terminal threonine -amino group as a
nucleophile to attack the deoxyribose C-1` in the glycosylase step. The
amino acid Glu
is crucial for this attack and for the
subsequent lyase
reaction(17, 18, 20, 21) . The
consequence of this mechanism is the formation of an imino enzyme-DNA
intermediate as the product of the glycosylase step (Fig. 1C). This intermediate may be hydrolyzed,
resulting in formation of an abasic site (Fig. 1D), or
undergo a
-elimination reaction, resulting in a lyase scission of
the sugar-phosphate DNA backbone (Fig. 1E). The ratio
of the rates of these two reaction legs would be responsible for the
observed pH dependence of the relative yields of abasic sites and
single strand breaks. Additional data to support the proposed mechanism
of endonuclease V have recently been obtained by isolation of a stable
covalent enzyme-DNA complex after reduction of the intermediate with
sodium borohydride (NaBH
)(19) .
Previous studies
have also shown that E. coli endonuclease III cleaves the
sugar-phosphate DNA backbone on the 3` side of an AP site via a
-elimination reaction rather than by a phosphotransfer
(endonuclease-type) activity(23, 24, 25) .
Furthermore, the E. coli FPG protein has been reported to cut
the DNA sugar-phosphate backbone by a
,
or
-elimination
reaction(26) .
As a generalization of these results, a hypothesis has been proposed (27) that the initial action of all DNA glycosylases proceeds by a nucleophilic attack at the sugar of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. In this hypothesis, the DNA glycosylases, which carry out an accompanying AP lyase reaction, are proposed to use an amino group as the attacking nucleophile, resulting in an imino enzyme-DNA intermediate (Fig. 1C). Simple glycosylases, lacking this concomitant AP lyase activity, are proposed to use some nucleophile from the medium, e.g. an activated water molecule, to promote catalysis (Fig. 1B). In this second case, no covalent enzyme-DNA intermediate is predicted to occur.
An
experimental consequence of this hypothesis is that it should be
possible to trap the putative lyase covalent intermediate by reduction
of the imino intermediate with NaBH. This would result in a
covalent enzyme-DNA complex which should be resolvable by denaturing
polyacrylamide gel electrophoresis. Enzyme inhibition and formation of
stable covalent complexes are the primary end points of the protocol
used in this study.
Figure 2:
Concentration dependence and inhibition by
NaBH of E. coli endonuclease III incising a
37-base pair oligonucleotide duplex that contains a site-specific
dihydrouracil. The oligonucleotide strand containing dihydrouracil was
5`-end-labeled with
P. The substrate (0.06 pmol) was
reacted with endonuclease III, and the products were separated by 15%
denaturing PAGE. Lane 1, oligonucleotide markers. Lane
2, 37-mer substrate alone. Lanes 3-6, reaction of
the substrate with decreasing amounts of the enzyme (0.28, 0.06, 0.01,
and 0.002 pmol from lane 3 to lane 6, respectively). Lanes 7-10 are similar to lanes 3-6,
except that the reactions were performed in the presence of 100 mM NaBH
.
Figure 3:
Probing for an E. coli endonuclease III-DNA substrate covalent complex by NaBH reduction. The dihydrouracil containing substrate (0.06 pmol) was
reacted with the enzyme in the presence of 100 mM NaCl or
NaBH
, and the products were subjected to 15% SDS-PAGE. Lane 1, substrate alone. Lane 2, reaction with 5.6
pmol of enzyme in the presence of NaCl. Lanes 3-6 resulted from the reactions of the substrate with decreasing
enzyme amounts (2.8, 0.56, 0.11, and 0.02 pmol from lane 3 to lane 6, respectively) in the presence of
NaBH
.
E. coli formamidopyrimidine
(Fapy) glycosylase (FPG) has been shown to recognize and remove
ring-opened purines (Fapy bases) and 8-oxo-deoxyguanine in duplex DNA.
FPG has been proposed to work via a concerted glycosylase
,
-elimination process (reviewed in (30) ).
Previously, it had been proposed that an amino group on the enzyme was
involved in a nucleophilic attack on the C-1` of 8-oxo-dG, with
formation of a Schiff base intermediate between the enzyme and its
substrate(30) . The FPG substrate used in this study, a 49-base
oligonucleotide duplex containing a unique 8-oxo-deoxyguanine at
position 21, was synthesized as described under ``Experimental
Procedures.'' FPG was found to cleave this substrate at the
damaged site in a dose-dependent manner as revealed by analysis of the
products through denaturing PAGE (Fig. 4, lanes
3-6). The nicking activity of FPG was completely inhibited
by the presence of NaBH
in the reaction mixture (Fig. 4, lanes 8-11). The
P-labeled
DNA substrates were trapped in the wells in a dose-dependent manner,
suggesting the formation of an enzyme-DNA covalent complex between FPG
and its substrate. Further analysis demonstrated that a shifted band
was formed in the FPG reactions in the presence of NaBH
,
even when the reaction products were subjected to SDS-PAGE (Fig. 5), indicating the existence of a covalent FPG
DNA
complex. Quantitative analysis using BioImage software revealed that,
at two different FPG concentrations, the percentages of the FPG cleaved
products in the absence of NaBH
(33.7 and 12.0% cleaved, as
shown in Fig. 4, lanes 4 and 5) were similar
to the percentages of the observed covalently trapped complex using the
same two FPG concentrations in the presence of NaBH
(30.0
and 9.0% trapped, as shown in Fig. 5, lanes 7 and 8). This suggests that the breakage of the DNA backbone occurs
via formation of a covalent enzyme-DNA complex. In contrast to what was
observed for endonuclease III, the minimum ratio of enzyme to DNA that
gave easily detectable gel shifted DNAs was 20 to 1. However, longer
exposure of these gels revealed gel shifted DNAs at mole ratios of 4 to
1. It has not been determined whether this might be due to a high
percentage of inactive enzyme in this preparation.
Figure 4:
Concentration dependence and inhibition by
NaBH of E. coli FPG incising a 49-base pair
oligonucleotide duplex that contains a site-specific
8-oxo-deoxyguanine. The oligonucleotide strand (approximately 0.05
pmol) containing 8-oxo-deoxyguanine at position 21 was 5`-end-labeled
with
P. The substrate was reacted with FPG in the presence
of 100 mM NaCl or 100 mM NaBH
, and the
products were separated by 15% denaturing PAGE. Lane 1,
markers. Lane 2, substrate alone. Lanes 3-6,
reactions contained decreasing amounts of FPG (9.9, 5.0, 1.0, and 0.5
pmol from lane 3 to lane 6, respectively) in the
presence of 100 mM NaCl. Lanes 7-11 are similar
to lanes 2-6 except that the reactions were carried out
in the presence of 100 mM
NaBH
.
Figure 5:
Probing for an FPG-substrate covalent
complex by NaBH reduction. The substrate (approximately
0.05 pmol) was reacted with FPG in the presence of 100 mM NaCl
or NaBH
, and the products were subjected to 15% SDS-PAGE. Lane 1, substrate alone. Lanes 2-6, reactions
of the substrate with decreasing FPG amounts (5.0, 1.0, 0.2, 0.04, and
0.008 pmol from lane 2 to lane 6, respectively) in
the presence of NaCl. Lanes 7-11 are similar to lanes 2-6 except that the reactions were carried out in
the presence of NaBH
.
Figure 6:
Concentration dependence and inhibition by
NaBH of E. coli MutY incising a 50-base pair
oligonucleotide duplex that contains a site-specific A/G mismatch. The
oligonucleotide strand containing the adenine of an A/G mismatch at
position 21 was 5`-end-labeled with
P. The substrate
(0.025 pmol) was reacted with MutY in the presence of 100 mM NaCl or NaBH
, followed by ± piperidine
treatment. The products were separated by 15% denaturing PAGE. A, lane 1 is the 50-mer substrate alone. Lanes
2-6 resulted from reactions containing decreasing amounts of
MutY (0.56, 0.11, 0.02, 0.005, and 0.001 pmol from lane 2 to lane 6, respectively). Lanes 7-12 are similar
to lanes 1-6 except that the reactions were followed by
piperidine treatment. B, lane 1, markers. Lane 2 resulted from the reaction of a 50-mer A/T control with MutY
followed by piperidine treatment. Lane 3 is substrate alone. Lane 4 resulted from the reaction of the substrate with MutY
(0.56 pmol). Lanes 5 and 6 resulted from the reaction
of the substrate with MutY (0.56 pmol in lane 5, 0.11 pmol in lane 6) in the presence of NaCl, followed by piperidine
treatment. Lanes 7 and 8 are similar to lanes 5 and 6, except that the reactions were carried out in the
presence of NaBH
.
Figure 7:
Probing for an enzyme-DNA substrate
covalent complex by NaBH treatment of E. coli MutY
reactions. MutY was incubated with its substrate (0.05 pmol) in the
presence of 100 mM NaCl or NaBH
, and the products
were subjected to 15% SDS-PAGE. Lane 1, substrate alone. Lane 2, reaction of the enzyme (2.8 pmol) with its substrate
in the presence of NaCl. Lanes 3-5, reactions with
decreasing enzyme amounts (2.82, 0.56, and 0.11 pmol from lane 3 to lane 5, respectively) in the presence of
NaBH
.
E. coli uracil-DNA glycosylase (UDG) is
responsible for the removal of uracil from DNA(11) . Uracil can
arise in double-stranded DNA by incorporation of dUTP in place of dTTP
or by deamination of cytosine. UDG is strictly a simple glycosylase,
lacking detectable AP lyase activity. E. coli UDG was shown to
catalyze the removal of uracil in a dose-dependent fashion (Fig. 8A). UDG was also inhibited by NaBH,
and, as was the case for MutY, there was no evidence for trapping of a
covalent dead-end intermediate (Fig. 8B). These
denaturing gel electrophoresis data were confirmed when no shifted band
was observed in nondenaturing PAGE (Fig. 9). Although the
highest mole ratio of enzyme to DNA shown in Fig. 9was 5 to 1,
additional experiments have confirmed that no covalent intermediates
are detectable at enzyme to DNA ratios as high as 25 to 1 (data not
shown). Therefore, there is no evidence for the formation of a covalent
enzyme-substrate intermediate in the case of UDG.
Figure 8:
Concentration dependence and inhibition by
NaBH of E. coli UDG incising a 49-base pair
oligonucleotide duplex that contains a site-specific uracil. The
oligonucleotide strand containing the uracil at position 21 was
5`-end-labeled with
P. The substrate (0.026 pmol) was
reacted with UDG in the presence of 100 mM NaCl or
NaBH
, followed by ± piperidine treatment, and the
products were separated by 15% denaturing PAGE. A, lane 1 is the 49-mer substrate alone. Lanes 2-6 resulted
from reactions containing decreasing amounts of UDG (0.12, 0.025,
0.005, 0.001, and 0.0002 pmol from lane 2 to lane 6,
respectively). Lanes 7-12 are similar to lanes
1-6 except that the reactions were followed by piperidine
treatment. B, lane 1, markers. Lane 2 resulted from the reaction of a 49-mer control (without uracil)
with UDG followed by piperidine treatment. Lane 3 is substrate alone. Lane 4 resulted from the reaction of the substrate with UDG
(0.12 pmol). Lanes 5 and 6 resulted from the reaction
of the substrate with UDG (0.12 pmol in lane 5, 0.025 pmol in lane 6) in the presence of NaCl, followed by piperidine
treatment. Lanes 7 and 8 are similar to lanes 5 and 6, except that the reactions were carried out in the
presence of NaBH
.
Figure 9:
Probing for an enzyme-DNA substrate
covalent complex by NaBH treatment of E. coli UDG
reactions. UDG was incubated with its substrate (0.03 pmol) in the
presence of 100 mM NaCl or NaBH
, and the products
were subjected to 10% nondenaturing PAGE. Lane 1, substrate
alone. Lane 2, reaction of the enzyme with its substrate in
the presence of NaCl. Lanes 3-5, reactions with
decreasing enzyme amounts (0.12, 0.03, and 0.005 pmol from lane 3 to lane 5, respectively) in the presence of
NaBH
.
Mammalian MPGs
have been reported to have a broad substrate specificity, as does the E. coli 3-methyl adenine DNA glycosylase II, the product of
the alkA gene. Substrates include all three N-alkylpurines but
not O-2-alkylpyrimidines in DNA(32) . Recent studies
have established that both AlkA and mammalian MPGs are capable of
removing etheno adducts of A, G, and C from DNA(33) . In the
current study, a slow cleavage of the sugar-phosphate DNA backbone of
an etheno-A-containing substrate was observed for mouse MPG, although
this rate was at least 2 orders of magnitude slower than the rate of
the glycosylase step (Fig. 10). Whether this result reflects an
intrinsic enzyme activity or a minor contamination is not clear.
Consistent with a recent report(32) ,
1-N-ethenoadenine-containing DNA was shown to be a
specific substrate for mouse MPG (Fig. 10). MPG was also
observed to be completely inhibited by NaBH
(Fig. 11), and like UDG and MutY, there was no evidence
for the existence of a covalent MPG enzyme-substrate intermediate, even
at enzyme to DNA ratios as high as 180 to 1 (Fig. 11, lanes
3-5). Again, the absence of the formation of gel shifted
DNAs is indicative of a nucleophilic attack by some other group than a
primary amine.
Figure 10:
Concentration dependence of mouse MPG
incising a 25-base pair oligonucleotide duplex that contains a
site-specific ethenoadenine adduct. The oligonucleotide strand
containing the ethenoadenine at position 6 was 5`-end-labeled with P. The substrate (0.05 pmol) was reacted with MPG,
followed by ± piperidine treatment, and the products were
separated by 20% denaturing PAGE. Lane 1, markers. Lane
2, 25-mer substrate alone. Lanes 3-7 resulted from
reactions containing decreasing amounts of MPG (8.9, 1.8, 0.36, 0.07,
and 0.01 pmol from lane 3 to lane 7, respectively). Lanes 8-13 are similar to lanes 2-7,
except that the reactions were followed by piperidine
treatment.
Figure 11:
Probing for an enzyme-DNA substrate
covalent complex by NaBH treatment of mouse MPG reactions.
MPG was incubated with its substrate (0.05 pmol) in the presence of 100
mM NaCl or NaBH
, and the products were subjected
to 10% nondenaturing PAGE. Lane 1, substrate alone. Lane
2, reaction of the enzyme with its substrate in the presence of
NaCl. Lanes 3-5, reactions with decreasing enzyme
amounts (8.9, 1.8, and 0.36 pmol from lane 3 to lane
5) in the presence of
NaBH
.
In summary, the two glycosylase/AP lyase enzymes
tested (endonuclease III and FPG from E. coli) could be
covalently trapped to their DNA substrates by treatment with
NaBH, whereas the three simple glycosylases (E. coli UDG and MutY and mouse MPG) could not.
Fig. 1is a schematic representation of our current hypothesis describing catalysis for both DNA glycosylases and DNA glycosylase/AP lyases. A provides an overview of the basic features that are necessary to initiate repair at either damaged bases or abasic sites. The initial interaction between the enzyme and DNA is a random collision process (A, step 1), and the successful binding to nontarget DNA generally involves electrostatic interactions. In order to locate target sites (damaged bases (A, step 2) or abasic sites (A, step 3)), many of the DNA glycosylases and glycosylase/AP lyases translocate along DNA through a random, one-dimensional process, referred to as scanning or facilitated diffusion (reviewed in (2) ).
Upon binding to a damaged
base, the simple DNA glycosylases catalyze the release of the damaged
or inappropriate base by nucleophilic attack by an activated water
molecule (A, step 4), leading to an abasic site and
enzyme release. This model predicts that, due to the nature of the
attacking nucleophile (B), no covalent intermediate should be
trappable by NaBH in the case of the simple glycosylases.
Data presented in Fig. 6-11 for MutY, UDG, and MPG all
support this prediction. However, direct evidence for this mechanism
has been obtained for UDG from herpes simplex virus type-1, as revealed
by x-ray crystallographic analyses of the enzyme in the presence and
absence of DNA(34) . The crystal structure model shows a well
ordered water molecule ideally positioned, and interacting with
appropriate protein side chains, to serve as the nucleophile for the
flipped-out (extrahelical) uracil base. The crystal structure and
mutational analysis of human UDG also indicate that His
may abstract a proton from a water molecule, creating an
OH
nucleophile that would then carry out the attack
at C-1`(35) . These co-crystal structures provide strong
support, in the case of UDG, for our mechanistic model of simple
glycosylases.
For the DNA glycosylase/AP lyases, the progress of the
reaction scheme is more complex (A, steps 5-7) and can
be illustrated by conclusions drawn from previous studies on
endonuclease V. However, all of the data accumulated for FPG and
endonuclease III described in this paper (Fig. 2-5) are in
complete agreement with the endonuclease V studies. The catalytic
activities of T4 endonuclease V have been extensively studied (reviewed
in (27) and (28) ) and are schematically diagrammed in Fig. 1(A, steps 5-7, and C-E). The mechanism first proposed by Schrock and Lloyd (15) proceeds via a nucleophilic attack by the N-terminal
-amino group of the enzyme upon the C-1` carbon of the sugar on
the 5`-most nucleoside in the cyclobutane pyrimidine dimer substrate. A
protonated Schiff base intermediate is subsequently formed involving
the N-terminal
-amino group and the sugar (A, step
5, and C). The fate of this intermediate is to be
hydrolyzed either before (A, step 6, and D)
or after the DNA undergoes a
-elimination reaction (A, step
7, and E). This reaction results in either an AP site (A, step 6, and D) or scission of the
sugar-phosphate backbone (A, step 7, and E),
respectively. The propensity of the enzyme to catalyze a single strand
break can be thought of as a competition between reaction E and reverse reaction D, which should depend on the pH of
the medium. The rationalized mechanism of action for the DNA
glycosylases tested in this study describes all DNA glycosylases as
catalyzing their N-glycosylase reactions via nucleophilic
attack on the sugar of their damaged base substrates (Fig. 1A). The bifurcation between the glycosylase/AP
lyases and the simple glycosylases lies with the chemical nature of the
attacking nucleophile. The glycosylase/AP lyases are hypothesized to
utilize primary amines in that role and to form protonated Schiff base
intermediates, such as that described for T4 endonuclease V. The simple
glycosylases, on the other hand, are proposed to use nucleophiles from
the medium for nucleophilic attack and not to form covalent enzyme-DNA
intermediates.
The ability of endonuclease III and FPG to form
covalent enzyme-substrate complexes in the presence of NaBH and substrate DNA ( Fig. 3and Fig. 5) provides
substantial support for the hypothesis that imino intermediates are
involved in the catalytic mechanism of these enzymes. As predicted by
our hypothesis, no enzyme-substrate complexes were detected in the
simple glycosylases MutY (Fig. 7), UDG (Fig. 9), or MPG (Fig. 11), indicating that an imino intermediate is not likely
to be involved in these enzymes' catalytic mechanisms. We suggest
that the
-elimination reaction does not occur in the reactions of
the simple glycosylases because the electron withdrawal effects of the
protonated Schiff base intermediate (Fig. 1B) are
required for the 2`-hydrogen to be labilized.
Further evidence to
support this hypothesis has come from other reports. The synthetic
tripeptide, Lys-Trp-Lys, has been proposed to cleave DNA containing an
AP site through a Schiff base intermediate formed by nucleophilic
attack of one of the lysine -amino groups on the aldehydic carbon
(C-1`) of the abasic site deoxyribose(36, 37) . This
hypothesis has been supported by trapping the Lys-Trp-Lys-DNA
intermediate by reduction with sodium cyanoborohydride. Also, the
mechanism of cleavage at an AP site by endonuclease III has previously
been proposed by Kow and Wallace (25) to proceed by a
-elimination mechanism. Similarly, the reaction of FPG has been
previously proposed to proceed through a
,
-elimination
mechanism by way of the formation of a Schiff base
intermediate(30) . In addition, proton abstraction during
-elimination has been shown to occur by a syn stereochemical course for both endonuclease III and T4
endonuclease V. This is opposite to the stereochemistry observed for
hydroxide ion-mediated backbone cleavage at an abasic site. In an
additional mechanistic analogy between the reactions of endonucleases
III and V, the crystal structure of endonuclease III (38) shows
that Asp
is located close in space to Lys
,
suggesting that Asp
may play a similar role to that of
the Glu
of endonuclease V in stabilizing the charge of the
imino intermediate(18) .
It should be emphasized that all data presented in this publication are consistent with our original hypothesis(27) . An understanding of the unified mechanism for DNA repair glycosylases will facilitate the identification of active site residues for other enzymes of this class, and will, hopefully, pave the way for an eventual understanding of the structure-function relationships within this important class of enzymes.