N-glycosyl bond formation catalyzed by human alkyladenine DNA glycosylase

Article abstract of DOI:10.1021/bi101380d

The removal of damaged bases by DNA glycosylases is thought to be effectively irreversible, because of an overall equilibrium that favors hydrolysis over synthesis of the N-glycosyl bond. Surprisingly, human alkyladenine DNA glycosylase (AAG) can make damaged DNA by catalyzing formation of an N-glycosyl bond between 1,N⁶-ethenoadenine (εA) and abasic DNA. We attribute the ready reversibility of this glycosylase reaction to the exceptionally tight binding and slow subsequent hydrolysis of DNA containing an εA lesion. In principle, reversibility could provide a mechanism for direct reversal of base damage by a DNA glycosylase, allowing the glycosylase to bypass the rest of the base excision repair pathway.

Full text of DOI:10.1021/bi101380d

9024 Biochemistry 2010, 49, 9024–9026
DOI: 10.1021/bi101380d
N-Glycosyl Bond Formation Catalyzed by Human Alkyladenine
DNA Glycosylase†
Suzanne J. Admiraal and Patrick J. O’Brien*
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-5606, United States
Received August 26, 2010; Revised Manuscript Received September 27, 2010
Scheme 1: Duplex DNA Oligonucleotides Used in This Study
ABSTRACT: The removal of damaged bases by DNA glyco-
sylases is thought to be effectively irreversible, because of
an overall equilibrium that favors hydrolysis over synthesis
of the N-glycosyl bond. Surprisingly, human alkyladenine
DNA glycosylase (AAG) can make damaged DNA by cata-
lyzing formation of an N-glycosyl bond between 1,N6-
ethenoadenine (εA) and abasic DNA. We attribute the ready
reversibility of this glycosylase reaction to the exceptionally
tight binding and slow subsequent hydrolysis of DNA
containing an εA lesion. In principle, reversibility could
provide a mechanism for direct reversal of base damage by
a DNA glycosylase, allowing the glycosylase to bypass the
rest of the base excision repair pathway.
(∼70%), conversion of εdA-DNA to ab-DNA [Figure 1A (0)],
whereas a slower reaction that proceeded to completion was
expected for simple product inhibition of AAG by εA.
We tested the possibility that εA reacts reversibly with ab-DNA
to regenerate εdA-DNA by adding free εA to AAG ab-DNA.
3
Reactions containing 0.6 mM εA that were initiated with either
AAG ab-DNA or AAG εdA-DNA proceeded to the same end
3
3
point of ∼0.3 [Figure 1A (open symbols)], which shows the same
equilibrium between AAG-bound species being approached
from opposite directions. To verify that the plateau reached in
The equilibria for hydrolysis of the N-glycosyl bonds of
nucleotides and nucleosides favor their nucleobase and ribose
products (1, 2). Indeed, de novo and salvage pathways for
nucleotide biosynthesis typically use 5-phosphoribosyl-R-pyro-
phosphate (PRPP) as a substrate for N-glycosyl bond formation,
as its utilization is driven by the subsequent hydrolysis of released
PPi. Base excision repair DNA glycosylases hydrolyze the analogous
N-glycosyl bond between a damaged base and C1 of a deoxyri-
bosyl moiety, giving rise to a free base and an abasic site within
DNA (3). Although the overall equilibrium for this reaction favors
hydrolysis, human alkyladenine DNA glycosylase (AAG) un-
expectedly synthesizes 1,N6-ethenodeoxyadenosine (εdA) within
DNA when supplied with 1,N6-ethenoadenine (εA) and abasic
DNA. The internal equilibrium constant (Kint) for this enzymatic
transformation is near unity.
Mutagenic εdA lesions form when adenine bases in DNA react
with certain lipid peroxidation products or pollutants (4, 5). AAG
is the only human glycosylase known to excise εA from εdA-
containing DNA, and its mechanism has been proposed to
include fast two-step DNA binding, rate-limiting N-glycosyl
bond hydrolysis, and rapid dissociation of εA (6). We explored
possible inhibition of the AAG reaction by its εA product by
the AAG ab-DNA reaction represented the equilibrium between
3
AAG ab-DNA and newly synthesized AAG εdA-DNA, we
3
3
tried to perturb it predictably. We first increased the AAG
3
ab-DNA concentration while maintaining the εA concentration
at 0.6 mM and followed the conversion of ab-DNA to εdA-DNA
for 100 min, until the expected end point of ∼0.3 had been
attained. The reaction mixture was then diluted 25-fold, resulting
in a mixture containing only 24 μM εA, and the decay of the
putative εdA-DNA was followed to its new equilibrium position
(Figure 1B). The rates of decay of the putative εdA-DNA and
authentic εdA-DNA were the same within error (Figure 1B,
inset), consistent with the model in which AAG converts ab-DNA
and εA into εdA-DNA via direct reversal of the glycosylase
hydrolysis step (Figure 2, ksyn).
We next investigated the dependence of the synthesis reaction
on the concentration of εA. As shown in Figure 3A, reactions of
AAG ab-DNA with εA proceeded with faster initial rates to
3
higher end points as the εA concentration increased. The reaction
end points are plotted as a function of εA concentration in
Figure 3B, and saturation with εA is observed with a K1/2 of
0.9 mM. At saturating concentrations of εA, the fractional concen-
adding the free base to AAG εdA-DNA, where εdA-DNA is a
3
25 bp oligonucleotide that contains a centrally located εdA
(Scheme 1). The single-turnover rate constant of 0.04 min-1
obtained for the AAG-catalyzed hydrolysis of εdA-DNA to ab-
DNA in the absence of added εA matched the value of khyd
measured previously [Figure 1A (9)] (6). Addition of 0.6 mM εA
to an otherwise identical reaction resulted in faster, but only partial
tration of AAG εdA-DNA is 0.7 (the maximum end point), and
3
the fractional concentration of AAG ab-DNA εA is corre-
3
3
spondingly 0.3, indicating that εdA-DNA is more stable than
its hydrolysis products when bound to AAG. The internal
equilibrium constant for these AAG-bound species is the ratio
of their fractional concentrations (Kint = 0.3/0.7 = 0.4). Kint is
also equivalent to khyd/ksyn, the ratio of the respective rate
constants for formation of AAG ab-DNA εA and AAG εdA-
†This work was supported in part by National Institutes of Health
Grant CA122254 to P.J.O.
*To whom correspondence should be addressed. Telephone: (734)
647-5821. Fax: (734) 764-3509. E-mail: pjobrien@umich.edu.
3
3
3
DNA from one another, so a value of 0.1 min-1 for ksyn can be
calculated from Kint and the reported value of 0.04 min-1 for
r
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Rapid Report
Biochemistry, Vol. 49, No. 42, 2010 9025
F
IGURE 1: AAG synthesizes εdA-DNA from εA and ab-DNA. (A) The fraction of total DNA present as εdA-DNA in quenched samples
from single-turnover reactions of AAG with εdA-DNA or ab-DNA was determined using a gel-based glycosylase assay (see the
Supporting Information). Error bars indicate the standard deviation from the mean for at least two independent determinations, and
lines are the best fits to single-exponential equations. (B) A single-turnover reaction containing 1.25 μM AAG ab-DNA and 0.6 mM εA
3
was followed to completion and then diluted 25-fold with buffer, resulting in a postdilution reaction of 50 nM AAG DNA and 24 μM εA
that was followed to its new equilibrium position. The inset shows the normalized postdilution reaction alongside a single-turnover
3
reference reaction of 50 nM AAG εdA-DNA. Although best fits of both inset reactions to single-exponential equations give kobs values
3
of 0.04 min-1, representing the rate constant for single-turnover excision of εA from εdA-DNA (khyd), the postdilution reaction does not
reach an end point of zero because of the residual εA that it contains. All reactions were performed at 25 °C and pH 6.5, and εdA-DNA
reactions were corrected to account for the small percentage of ring-opened substrate that is refractory to AAG reaction (see the Supporting
Information).
Interestingly, the structure of AAG crystallized in the pre-
sence of an εdA-containing DNA substrate revealed an intact
N-glycosyl bond (10). Although the authors proposed that the high
concentration of MgCl2 present during crystallization may have
inhibited the glycosylase activity of AAG, our results raise the
possibility that the substrate may have instead persisted because
of the internal equilibrium that favors synthesis of εdA-containing
DNA over its hydrolysis by AAG. The crystal structure shows
εdA flipped into the AAG active site, securely held by aromatic
stacking interactions and a hydrogen bond between the backbone
amide of His-136 and N6 of the nucleobase. This snug fit helps to
account for the observed tight binding of εdA-containing DNA
by AAG (6, 11, 12) and the highly favorable equilibrium for
flipping εdA into the active site [Kflip ∼ 1300 (6)]. The unusual
stability of εdA-DNA when bound to AAG, which enabled us to
detect its synthesis and to measure the internal equilibrium
constant for its hydrolysis, presumably arises from its tight
binding to the active site coupled with slow subsequent hydro-
lysis of its N-glycosyl bond. In contrast, human uracil DNA
glycosylase (UDG) has been crystallized bound to its uracil and
abasic DNA products (13, 14), consistent with an internal
equilibrium for UDG that favors hydrolysis of uracil-containing
DNA (15).
F
AAG under single-turnover conditions, where all the DNA present is
bound to AAG.
IGURE 2: Model for reversible excision of εA from εdA-DNA by
khyd [Figure 1A (6)]. The best global fit of the data in Figure 3A
to the model in Figure 2 gives slightly different values of 0.2 for
Kint and 0.2 min-1 for ksyn (see the Supporting Information), so
we report an average value of 0.3 ( 0.1 for Kint
.
To the best of our knowledge, the conversion of ab-DNA and
εA to εdA-DNA by AAG is the first example of N-glycosyl
bond synthesis by a DNA glycosylase. We assessed the general-
ity of this reverse reaction by investigating several related
reactions. AAG did not insert either adenine or hypoxanthine
into ab-DNA, despite extended incubation times with milli-
molar concentrations of each free base, nor was any reaction
observed when a different DNA glycosylase, 3-methyladenine
DNA glycosylase II (AlkA), was incubated with ab-DNA and
εA (data not shown). Thus, the ability of AAG to stably
synthesize εdA-DNA appears to be unusual. A related enzyme
activity that inserts purines into depurinated DNA has been
reported previously (7, 8), and kinetic isotope effects for the
DNA glycosylase MutY are consistent with a dissociative
mechanism in which reversible C-N bond cleavage precedes
irreversible nucleophilic attack by water (9).
As described above, results from reactions at saturating εA
concentrations allowed determination of a value of 0.3 for Kint
,
the equilibrium constant for hydrolysis of the N-glycosyl bond of
AAG-bound εdA-DNA. Kint values near unity are considered to
be optimal for enzymes that operate reversibly (16). While it
stands to reason that certain metabolic enzymes that act at the
N-glycosyl bond may benefit from reversibility (e.g., nucleosidases
and nucleoside phosphorylases), synthesis of εdA-containing
DNA by AAG is expected to be negligible under typical condi-
tions, because of low cellular concentrations of abasic DNA and εA.
In principle, however, ready reversibility provides a potential
mechanism for direct repair by DNA glycosylases, in which
excision and dissociation of an aberrant base from DNA are

9026 Biochemistry, Vol. 49, No. 42, 2010
Admiraal and O’Brien
ACKNOWLEDGMENT
We thank Tom Ellenberger for help with initial experiments
and members of the O’Brien lab for useful discussions and
comments on the manuscript.
SUPPORTING INFORMATION AVAILABLE
Detailed experimental procedures and supplementary figures.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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