Mechanistic studies on histone catalyzed cleavage of apyrimidinic/apurinic sites in nucleosome core particles

Article abstract of DOI:10.1021/ja306858m

Duplex DNA containing an apurinic/apyrimidinic (AP) lesion undergoes cleavage significantly more rapidly in nucleosome core particles (NCPs) than it does when free. The mechanism of AP cleavage within NCPs was studied through independently generating lesions within them. AP mediated DNA cleavage within NCPs is initiated by DNA-protein cross-link (DPC_un) formation followed by β-elimination to give DPCs containing cleaved DNA (DPC _cl). Hydrolysis of DPC_cl produces a DNA single strand break (SSB). C2-dideuteration of AP showed that deprotonation from this position is involved in the rate-determining step. Experiments utilizing NCPs containing mutated histone H4 proteins indicated that lysine residues in the amino terminal tail are involved in both DPC formation and β-elimination steps. Lysines 16 and 20 seem to play a greater role in reacting with AP at superhelical location 1.5, but other amino acids (e.g., lysines 5, 8, and 12) compensate in their absence. The mechanism of rapid double strand breaks in bistranded, clustered AP lesions was studied by independently preparing reaction intermediates within model NCPs. A single strand break on one strand enhances the cleavage of a proximal AP on the opposite strand.

Full text of DOI:10.1021/ja306858m

Article
pubs.acs.org/JACS
Mechanistic Studies on Histone Catalyzed Cleavage of Apyrimidinic/
Apurinic Sites in Nucleosome Core Particles
Chuanzheng Zhou, Jonathan T. Sczepanski, and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: Duplex DNA containing an apurinic/apyrimi-
dinic (AP) lesion undergoes cleavage significantly more rapidly
in nucleosome core particles (NCPs) than it does when free.
The mechanism of AP cleavage within NCPs was studied
through independently generating lesions within them. AP
mediated DNA cleavage within NCPs is initiated by DNA−
protein cross-link (DPCun) formation followed by β-
elimination to give DPCs containing cleaved DNA (DPCcl). Hydrolysis of DPCcl produces a DNA single strand break
(SSB). C2-dideuteration of AP showed that deprotonation from this position is involved in the rate-determining step.
Experiments utilizing NCPs containing mutated histone H4 proteins indicated that lysine residues in the amino terminal tail are
involved in both DPC formation and β-elimination steps. Lysines 16 and 20 seem to play a greater role in reacting with AP at
superhelical location 1.5, but other amino acids (e.g., lysines 5, 8, and 12) compensate in their absence. The mechanism of rapid
double strand breaks in bistranded, clustered AP lesions was studied by independently preparing reaction intermediates within
model NCPs. A single strand break on one strand enhances the cleavage of a proximal AP on the opposite strand.
Scheme 1
INTRODUCTION
■
Apyrimidinic/apurinic sites (AP) are constantly produced in
DNA due to spontaneous hydrolysis of the nucleotides’
glycosidic bonds at rates sufficient to produce at least 10 000
of these lesions per cell per day.1 Nucleobase alkylation
significantly increases the rate of hydrolysis of the glycosidic
bond, and antitumor agents and carcinogens that alkylate
nucleobases such as leinamycin and benzopyrene epoxides
produce AP sites with varying ease following initial reaction
with DNA.2,3 AP sites are also formed as intermediates during
base excision repair by enzymes such as uracil DNA
glycosylase.4 AP sites are alkali-labile lesions but undergo
strand scission slowly in free DNA at physiological pH.5
Recently, we reported that the ∼3 week half-life of an intact AP
site in free DNA is decreased as much as 60-fold in a
nucleosome core particle (NCP) composed of the α-satellite
DNA sequence and that persistent DNA−protein cross-links
(DPCs) are formed en route to strand breaks (Scheme 1).6
Herein, we examine the mechanism by which the histone
proteins in the nucleosome core particle catalyze strand scission
from AP.
amino group, nor is imine formation the only role for amines in
β-elimination.17,18 Binding can contribute significantly to the
lyase reactivity of a potential catalyst, as evidenced by
comparing the reactivity of two small molecules, the
Lys·Trp·Lys tripeptide and an amino acridine (intercalator)
equipped with an adenine to fill the void created by AP
formation in duplex DNA.18 The latter is effective at cleaving
DNA containing AP sites at 100 000-fold lower concentration
than the Lys·Trp·Lys tripeptide.
Lysine residues are critical in several processes involving AP
cleavage. For instance, lysines are responsible for Schiff base
formation during the β-elimination reactions characteristic of
the lyase activity of enzymes involved in base excision repair,
including Nth and DNA polymerase β.7−12 More recently, lyase
activity has been ascribed to proteins not previously thought to
play an active chemical role.13,14 Lysines are also components
of small peptidic mimics of lyase enzymes.8,15−17 However,
studies on the reactivity of AP sites with small molecules
indicate that Schiff base formation is not limited to the lysine ε-
Received: July 13, 2012
Published: September 28, 2012
© 2012 American Chemical Society
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The octameric core of histone proteins in nucleosome core
particles utilizes a large number of positively charged, basic
residues to bind the ∼146 base pairs of DNA that complete
∼1.7 turns as it wraps around the protein complex.19,20 In
addition, the histone proteins contain lysine and arginine rich,
flexible amino terminal tails that are potentially able to interact
with DNA at a variety of nucleotide positions. The ε-amino
groups of lysine residues in these tails are often post-
translationally modified via acetylation or methylation.21−24
This has significant biochemical consequences, but also alters
the amines’ chemical properties. Complexation of AP
containing DNA within NCPs provides a high effective molarity
of lysine and arginine amino acids that could catalyze strand
scission by β-elimination via general base catalysis, neutraliza-
tion of the phosphate monoester leaving group, and/or the
formation of Schiff bases (Scheme 1). The rapid cleavage of
DNA containing an oxidized abasic site produced by an
antitumor agent within chromatin provided the first glimpse
into the nucleosome’s ability to catalyze strand scission.25 We
have taken advantage of the ability to independently generate
lesions at defined sites in DNA, and technology for selectively
mutating the histone proteins that make up NCPs to investigate
this chemistry.6,26
wild type (wt) or mutated H4 proteins were reconstituted using
reported methods.31 Abasic sites were generated at two
positions in the region that is approximately 1.5 helical turns
from the dyad axis of the DNA (superhelical location (SHL)
1.5), a known hot spot for DNA binding/damaging
molecules.32,33 In each nucleosomal core particle, AP was
produced individually at position 89 on one strand and 3
nucleotides away on the opposite strand (AP207 when using the
146 bp α-satellite DNA or AP205 in the 145 bp 601 DNA)
(Figure 1).28 X-ray crystal structures of nucleosomes containing
RESULTS AND DISCUSSION
■
Design and Preparation of Nucleosome Core Particles
Containing AP. AP reactivity was examined in two 145−146
bp sequences, the α-satellite and “601” DNA.19,27 X-ray crystal
structures of NCPs composed of either DNA sequence have
been reported.19,20 The DNA sequences are positioned
remarkably similarly in the core particles, but the 601 sequence
was chosen for its strong binding.27 The DNA molecules were
prepared by enzymatic ligation of chemically synthesized
oligonucleotides and purified by denaturing polyacrylamide
gel electrophoresis (PAGE).6,26,28 The AP site was generated
after nucleosome core particle reconstitution from either 1
(photolysis) or dU (uracil DNA glycosylase, UDG) (Scheme
2). The photolytic precursor was employed in all kinetic
Figure 1. Independent generation of AP lesions in nucleosome core
particles. (A) X-ray crystal structure of the 601 NCP showing
nucleotide positions modified. From: PDB 3LZ0. (B) Local sequences
in 601 and α-satellite NCPs in which AP sites are incorporated. See
Supporting Information for entire DNA sequences and crystal
structure of α-satellite NCP.
α-satellite or 601 DNA with native nucleotides at the positions
where AP is introduced indicate that all of the lesions should
face toward the octameric core. Nucleosome core particles
containing AP on both strands (bistranded lesions) were
employed in experiments focused on double strand break
(DSB) formation. DNase I digestion demonstrated that the
rotational positioning of the nucleosome core particles was
unaffected by the modifications.28
Scheme 2
Isolated AP Reactivity in Nucleosome Core Particles
Composed of Wild Type Histone Proteins. Using
NaBH3CN as a trapping agent, we previously established that
AP cleavage in NCPs proceeds through DNA−protein cross-
links.6 The disappearance of AP89 and AP205 in NCPs
containing the 601 DNA sequence was followed over 24 h
and analyzed by sodium dodecyl sulfate (SDS) PAGE (Figure
2, Table 1).28 AP site disappearance fit well to first-order
kinetics (Table 1) and 3 products were observed. Two products
migrated considerably more slowly in the SDS-PAGE gel, in
which proteins are denatured and noncovalently bound DNA
dissociates from the proteins. The slower migrating products
were identified as DNA−protein cross-links based upon their
transformation into faster migrating products upon proteinase
K treatment. The products (Figure 3) were characterized
following their isolation from the corresponding bands from the
gel by the crush and soak method. The desalted materials were
experiments in order to eliminate enzyme activity as a
variable.29,30 UDG treatment on dU was used when
determining which histone protein(s) formed cross-links with
AP because of the necessity to radiolabel the 5′-phosphate of
the lesion.6,28
The histone octamer composed of a tetramer of dimers was
assembled from purified Xenopus proteins (H2A, H2B, H3, and
H4) that were overexpressed in Escherichia coli following
literature protocols.31 Purified mutant forms of the H4 protein
were obtained using Quickchange as previously described.26
Nucleosome core particles composed of octamers consisting of
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treated with proteinase K, and reanalyzed by denaturing PAGE
analysis.28 The migration of the proteinase K treated material in
a denaturing PAGE gel was compared to independently
prepared DNA containing intact AP that was either analyzed
directly or cleaved by treatment with NaOH. The DNA from
the more slowly migrating DNA−protein cross-link comigrated
with cleaved DNA in the denaturing PAGE gel, while the DNA
from the faster moving product comigrated with the intact AP
containing DNA.28 These DNA−protein cross-links were
concluded to contain cleaved (DPCcl) and uncleaved DNA
(DPCun), respectively. Similar analysis of the product that
migrates slightly more slowly than starting material in the SDS
PAGE gel indicated that these were single strand breaks (SSBs,
Figure 3).
The rate constants for AP disappearance in the 601 NCP
(Table 1) are within experimental error of those reported at the
comparable positions containing α-satellite DNA.6 The
acceleration in the NCPs compared to the free DNA is
approximately 3-fold greater in the 601 sequence than it was in
the α-satellite NCP. For instance, reactivity at AP89 is ∼110-
times greater in the 601 NCP than in the same sequence of free
DNA. However, the greater acceleration in the 601 core particle
compared to that containing α-satellite DNA is almost
completely due to slower rate constants for AP cleavage in
the free DNA, whose half-life is as long as 6 weeks.
Although the kinetics of AP disappearance were very similar,
the product distributions in the 601 (Figure 2) and α-satellite6
NCPs were different from one another. For example, the total
yield of DNA−protein cross-links (DPCs) was lower in the 601
nucleosome core particles at AP89 and AP205, and was
compensated for by higher single strand break (SSB) yields.
DPCs containing uncleaved DNA (DPCun) were not observed
after 1 h, which is slightly sooner than in the α-satellite NCP.
The DPCcl reach their maximum yield at 16 h and then
decrease, which is also faster than in α-satellite NCP. The
DPCs never rose above 10% at AP205, whereas they were the
major products in the α-satellite DNA containing nucleosome.
This indicates the hydrolysis of the putative Schiff base in
DPCcl (k3, Scheme 1), especially those involving AP205 is faster
in the 601 NCP.
NaBH3CN was used in situ to trap DPC intermediates by
reducing the CN to C−N. Incubating the 601 NCPs in the
presence of NaBH3CN (10 mM) significantly increases the
yield of DNA−protein cross-links at the expense of SSBs, but
the majority of DPCs trapped still consist of cleaved DNA
(DPCcl) (Figure 4). Higher concentrations of reducing agent
result in higher yields of DPCun at the further expense of single
strand break and DPCcl levels. Despite the changes in product
distribution, the rate constants for AP89 disappearance in the
NCP is unaffected by incubating with NaBH3CN, whereas the
half-life of AP205 is reduced by more than 40% in the presence
of the reducing agent (Table 1). These data affirm the previous
proposal that AP cleavage is catalyzed by the histone protein(s)
within the nucleosome core particle and proceeds through
DNA−protein cross-links.6 However, the different effects of
NaBH3CN on the rate constant of AP disappearance suggest
that the equilibrium corresponding to the initially formed Schiff
base (DPCun, Scheme 1) is more favorable for AP89 than for
AP205. Incubation of NCP core particle in the presence of
NaBH3CN traps DPCun and prevents its reversion to starting
material.
Figure 2. Reaction of (A) AP89 and (B) AP205 in a 601 nucleosome
core particle as a function of time.
Table 1. AP Reactivity as a Function of Position in 601
Nucleosome Core Particles and Free DNA at 37 °C
nucleosome core particle
free DNA
a
b
position
NaBH3CN
kDis (10−6 s−1
)
t1/2 (h)
t1/2 (h)
AP89
AP89
AP205
AP205
-
21.8 2.0
20.7 4.5
6.5 1.1
11.3 0.6
8.9 0.8
9.3 2.0
29.6 5.2
17.1 0.9
986
+
-
-
1008
-
+
a
Rate constants are averages std. dev. of at least 5 experiments, each
b
consisting of 3 independent reactions. Data were for a single
experiment consisting of 3 independent reactions in 100 mM NaCl, 1
mM EDTA, 10 mM HEPES (pH 7.5).
Figure 3. Representative SDS-PAGE gels (with and without
NaBH3CN) of products produced from AP89 in the 601 NCP.
The effect of C2-dideuteration on the rate of disappearance
of the lesion was determined for AP89 (Figure 5). A significant
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histone H3 increases to 28% at the expense of the H4 protein
(62%). Four lysines are present in the first 19 amino terminal
amino acids of the H4 protein, and a fifth is located at position
20 (Figure 6C). It is possible that either the α- and/or ε-amine
Figure 4. Reaction of AP89 in a 601 nucleosome core particle as a
function of time in the presence of NaBH3CN (10 mM).
Figure 6. Effect on histone H4 protein structure on half-life for
disappearance of AP89 in a 601 nucleosome core particle in the (A)
absence or (B) presence of NaBH3CN. (C) Amino terminal sequence
of histone H4 protein. Legend: wild type, wt; lysine, K; alanine, A.
of Lys20 in the 19 amino acid deletion mutant of histone H4 is
responsible for Schiff base formation. However, we were unable
to identify the amino acid(s) responsible for Schiff base
formation by mass spectrometry.
Figure 5. Effect of C2-dideuteraton on the rate of disappearance of
AP89 in a 601 nucleosome core particle.
Our inability to identify specific amino acids involved in
Schiff base formation using mass spectrometry led us to probe
the roles of individual lysines in the H4 protein by examining
the reactivity of AP89 in 601 nucleosome core particles
containing various mutated forms of this protein (Figure 6).
Lysines were targeted for mutation because they are most
typically associated with Schiff base formation. Histone H4 was
primarily responsible for DPC formation with AP89 in 601
NCPs containing wild type H4 and all mutant proteins studied
(vide infra).28 Substituting alanines for lysines 5, 8, and 12 had
no effect on the half-life of AP89 and replacing Lys16 decreased
the reactivity only slightly (Figure 6A). The Lys16,20Ala
double mutant showed the largest effect of any of the
substitution mutants, but AP89 reactivity was still only ∼2-
fold slower than in the NCP composed of wild type H4.
Deleting the 20 N-terminal amino acid H4 protein tail
containing 5 lysine residues had the largest effect, increasing
the half-life for AP89 disappearance ∼5-fold. However, even this
increase in the AP89 half-life accounts for only a portion of the
accelerated reactivity (>100-fold) of the abasic site within the
nucleosome core particle compared to free DNA (Table 1).
The mutant forms of histone H4 were also used to probe
Schiff base formation by trapping these intermediates with
NaBH3CN (Figure 6B). The half-life for AP89 disappearance
kinetic isotope effect (5.2 0.5) was measured. As in the case
of 2-deoxyribonolactone (L) cleavage, this indicated that C2-
deprotonation is involved in the rate determining step for AP89
disappearance within NCPs.26 C2-Dideuteration also reinforced
the notion that AP89 Schiff base hydrolysis (k−1, Scheme 1) is
rapid because despite reducing the rate constant for elimination
from DPCun only 3% of this cross-linked intermediate builds up
over 24 h.28 Overall, the observed rate constant for
disappearance of the AP site in the NCP (kDis) is expressed
in the context of a pre-equilibrium mechanism (eq 1, Scheme
1).
k1
k−1
kDis
=
k2
(1)
The Role of Lysines in the Histone H4 Tail on AP
Reactivity. Histone H4 is responsible for more than 95% and
88% of the DNA−protein cross-links at AP89 and AP207
,
respectively, in α-satellite DNA containing nucleosome core
particles.6 Removing the 19 N-terminal amino acids from H4
reduces the rate constant for AP disappearance 2- to 3 fold in
the respective NCPs but has no effect on the contribution of
cross-linking by histone H4 to AP89 relative to other proteins.
However, the amount of cross-linking between AP207 and
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increases less than 2-fold in any NCP containing mutant forms
of H4, including the 20 amino acid deletion. One interpretation
of these experiments is that different lysines in the H4 tails have
similar potency for reacting with AP89 to form Schiff bases.
Alternatively, AP89 would disappear with approximately the
same rate constant in the core particles containing mutant H4
proteins because reaction with NaBH3CN is the rate
determining step. We cannot rule this out.
The protein tail may also be involved in the β-elimination
step that transforms DPCun to DPCcl (Scheme 1). When the
NCP reconstituted using the H4 deletion mutant was incubated
in the presence of NaBH3CN, SSBs were largely replaced by
DNA−protein cross-links containing uncleaved DNA (DPCun,
Scheme 1, Table 2).28 Elimination cannot occur after reduction
Double Strand Break Formation from Bistranded
Lesions. Bistranded lesions contain two damage sites within
∼1.5 helical turns, are produced by γ-radiolysis, and are
believed to be especially deleterious to cells because they are
repaired more slowly than individual modifications or strand
breaks.35−41 The chemical reactivity of bistranded lesions in
nucleosome core particles had not been described prior to our
study involving α-satellite containing NCPs.6 Double strand
breaks resulted from DNA containing AP sites at positions 89
and 207 with no apparent induction period (Figure 7), even
though the overall rate constant for core particle disappearance
was at most slightly greater than when only a single lesion at
AP89 was present.
Table 2. Effect of mutations in histone H4 protein on the
distribution of DNA-protein cross-links at AP89 in a 601
nucleosome core particle in the presence of NaBH3CN
histone H4 protein
DPCun/DPCcl
Wild type
0.15
0.12
0.26
0.64
1.22
10.3
Lys5,8,12Ala
Lys16Ala
Lys20Ala
Lys16,20Ala
1−20 Deletion
by NaBH3CN. Recall, that DPCcl was the major product
trapped from NCP containing wild type proteins incubated
with NaBH3CN (Figure 4, Table 2). DPCcl was also the major
product when the Lys5,8,12Ala H4 mutant was incubated with
NaBH3CN. However, larger DPCun/DPCcl ratios were
observed when NCPs containing Lys16Ala and/or Lys20Ala
H4 mutants were incubated in the presence of reducing agent
(Table 2). These data are consistent with the hypothesis that
lysines 16 and 20 are more effective at inducing β-elimination at
AP89 than are lysines 5, 8, and 12, which is why NaBH3CN is
better able to compete with elimination when the former are
mutated to alanine.
Figure 7. Observed and calculated (Kintecus)42 reactivity of
bistranded AP in α-satellite DNA containing NCP.
We modeled single and double strand break growth in the
bistranded α-satellite DNA containing NCP (Figure 7), that
were previously measured by SDS gel electrophoresis following
proteinase K digestion, using a simplified kinetic scheme
(Scheme 3) that ignores the persistence of DNA−protein
Scheme 3
Despite the effect of various mutated proteins on AP89
reactivity, histone H4 remained the major species responsible
for Schiff base formation. Analysis of DPCs isolated from core
particles incubated in the presence of NaBH3CN in which the
5′-phosphate of AP89 was 32P-labeled revealed that histone H4
accounted for more than 95% of the cross-linked material in the
601 NCP composed of wild type proteins, alanine mutants, and
even in the NCP composed of the 20 amino acid deletion.6,28
The α-amino group of the N-terminal amine,34 which based
upon the crystal structure of the 601 NCP containing
undamaged DNA is located near position 89, is postulated to
be responsible for cross-linking in the deletion mutant.20
Upon the basis of these observations, we propose that lysines
are involved in the rate determining elimination step, as well as
in Schiff base formation. Lysines 16 and 20 seem to play a
greater role in reacting with AP89 but other amino acids (e.g.,
Lys5, 8, and 12) compensate in their absence. The flexibility of
the protein tail presumably enables multiple lysine residues to
react with AP89. The experiments utilizing the H4 deletion
protein suggest that other amino acids (e.g., arginines) and/or
the H3 histone tail may also contribute to the rate limiting
elimination step, especially when H4 is modified (Scheme 1),
but this has yet to be investigated.
cross-links following strand scission (DPCcl, Scheme 1).6,42 By
assuming that the rate constants for disappearance (cleavage) of
the first AP site in the bistranded core particle (k11 = 2.3 × 10−5
s−1, k21 = 6.1 × 10−6 s−1) were the same as in core particles in
which a single lesion was present, the program Kintecus42 did
an excellent job fitting DSB growth but the low SSB yields as a
function of time were less well reproduced (Figure 7). With
these caveats, the predicted rate constants for double strand
break formation from the SSBs (k12 = 3.9 × 10−4 s−1, k22 = 6.5
× 10−5 s−1) were 10−17 times faster than those for initial single
strand cleavage.
The 601 NCP containing abasic sites at positions 89 and 205
also yields double strand breaks rapidly with little if any
induction period (Figure 8). In contrast to the NCP containing
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Scheme 4
constants for cleavage of the second AP site are also predicted
to be much faster than the first.28
We suggested a template mechanism6 for the faster cleavage
of the second AP site in bistranded abasic sites. In this
mechanism (Scheme 4), DNA−protein cross-link formation at
one abasic site increases the effective molarity of other lysines
with respect to a neighboring lesion, resulting in more rapid
DSB formation than expected (k12 > k11 and k22 > k21, Scheme
3).
Figure 8. Reaction of bistranded abasic sites within a nucleosome core
particle as a function of time in which (A) the strand containing AP89
is 32P-labeled and (B) the strand containing AP205 is 32P-labeled.
To test the viability of the proposed template mechanism,
the kinetic effect of a DNA−protein cross-link on the reactivity
of a proximal AP site was independently examined by preparing
a model nucleosome core particle. We took advantage of the
ability to generate AP sites from two orthogonal precursors
(Scheme 2). Two 601 NCPs containing dU89 and 1205 or 189
and dU205 on opposite strands were prepared and treated with
UDG, followed by incubation in the presence of NaBH3CN to
trap the DNA−protein cross-links (Scheme 5). Using this
method, DPC-SSBs (Scheme 5) were formed in 50−70% yield
after 24 h. Following removal of the reducing agent, the AP site
on the opposite strand was generated photochemically. The
reactivity of the photochemically generated AP site in the
presence of the DPC-SSB (DPCcl) was monitored as a function
of time.28 A marked acceleration in AP reactivity was observed
in both NCPs. The half-life for AP89 decreased from ∼9 h
(Table 1) to less than 2 h (t1/2 = 113 32 min, k = 1.1 0.4 ×
10−4 s−1) when cleaved AP205 was trapped by a histone protein
(DPCcl, Scheme 1). The effect of DPC-SSB involving AP89 on
AP205 reactivity was even more striking. The half-life for AP205
in a NCP decreased from almost 30 h (Table 1) to less than 1 h
(t1/2 = 52 9 min, k = 2.3 0.4 × 10−4 s−1).
α-satellite DNA, the overall rate constant for disappearance of
the 601 NCP containing the bistranded lesion (strand
containing AP89 32P-labeled: kDis = 7.1 2.0 × 10−5 s−1, t1/2
= 2.7 0.8 h; strand containing AP205 32P-labeled: kDis = 6.1
0.9 × 10−5 s−1, t1/2 = 3.2 0.5 h) is more than twice as fast as
the core particle containing a single lesion at AP89.28 Single
strand breaks are not detected by SDS-PAGE, regardless of
which strand is 32P-labeled (Figure 8). The DNA−protein
cross-links containing cleaved DNA (DPC-SSB, Scheme 4)
yield (∼15%) reaches a maximum at ∼2 h and then declines to
∼4% over the course of the reaction. Proteins cross-linked to
the labeled strand are detectable in DSBs. The yields of protein
cross-linked DSBs (DPC-DSB) correlate with what is observed
in core particles containing a single abasic site (Figure 2). For
instance, the DPC-DSB yield is greater in the core particle in
which the strand containing AP89 is 32P-labeled (∼40%, Figure
8A), whereas the comparable product never reached 6% when
the strand containing AP205 is 32P-labeled (Figure 8B).
Kintecus analysis of the time dependence for SSBs and DSBs
in the 601 NCP containing AP89 and AP205 does not yield as
good a fit for product formation as was observed for the core
particle containing α-satellite DNA (Figure 7).28 Using the rate
constants for disappearance of AP89 or AP205 in core particles
containing isolated lesions may be a source of the poor fitting
because the bistranded lesion disappears more rapidly than do
the isolated abasic sites in the 601 NCP. Nonetheless, the rate
Experiments using the independently synthesized DNA−
protein cross-link suggest that the template mechanism can
result in faster cleavage of the second AP in bistranded basic
sites. However, it does not rule out an even simpler
explanation. For instance, given that the majority of the
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Scheme 5
Scheme 6
templated proteins would contain a strand break (DPC-SSB,
Scheme 5), we considered whether double strand break
formation is accelerated due to the presence of a SSB. Such
an effect could be rationalized by thinking of the AP site in the
vicinity of a strand break as being in a more single strand like
environment. Abasic lesions typically undergo strand scission in
single stranded DNA more readily than those in double
stranded DNA.43−45
To test the effect of a strand break on AP reactivity, core
particles containing 1 and a photolabile precursor (2, Scheme
6) to a direct strand break developed by Taylor were
independently synthesized.46 Substituting a direct strand
break for AP205 increased the reactivity of AP89 (t1/2 = 267 h)
by 3.7-fold in naked 601 DNA. Similarly, AP205 was 2.8-fold
more reactive (t1/2 = 364 h) when a strand break was present at
position 89. Greater AP reactivity in the presence of a strand
break was also observed in the nucleosome core particles. The
half-life for AP89 (t1/2 = 156 19 min, k = 7.4 0.9 × 10−5 s−1)
decreased more than 3-fold in the vicinity of a strand break
compared to when it was an isolated lesion within a NCP
(Table 1). The reactivity of AP205 (t1/2 = 96 8 min, k = 1.2
0.1 × 10−4 s−1) increased even more when part of a bistranded
lesion containing a strand break on the opposite strand at
position 89. AP205 reacted 20-times faster in a NCP containing
a proximal strand break on the opposite strand compared to
when it was the only lesion present.
the opposite strand cleavage through a template mechanism
(Scheme 4). However, mechanistic economy (Ockham’s razor)
requires that the increased rate of double-strand break
formation be ascribed to the formation of a single strand
break, which is common to both model systems examined
above (Schemes 5 and 6).47,48
Whether this is generally the case throughout nucleosome
core particles is uncertain and AP reactivity needs to be
examined at other superhelical locations. In addition, these
experiments do not account for the entire acceleration of abasic
site cleavage that is observed in nucleosomes compared to
naked DNA. Further studies are warranted to reveal the factors
responsible for this acceleration.
Regardless of the source of the large acceleration in abasic
site cleavage within nucleosome core particles, this study
further validates that the octameric histone core is more than a
structural scaffold. Histone catalyzed cleavage of (oxidized)
abasic site cleavage suggests that bistranded lesions containing
them are de facto double strand breaks. This chemistry may
help explain the high cytotoxicity of DNA damaging agents that
produce bistranded lesions.26,49,50
CONCLUSIONS
■
ASSOCIATED CONTENT
* Supporting Information
Histone cross-linking to one abasic site accelerates AP cleavage
on the opposite strand 5- to 30-fold. However, a proximal
single strand break also enhances AP cleavage on the opposite
strand 3−20 times within the NCP. In the case of bistranded,
clustered lesions containing two AP sites, DNA−protein cross-
link (DPCcl, Scheme 1) formation on one AP site may catalyze
■
S
Experimental procedures for all experiments, complete
sequences of all DNAs used to prepare nucleosome core
particles, representative autoradiograms and kinetic plots, NMR
and mass spectra of key compounds, and mass spectra of
16740
dx.doi.org/10.1021/ja306858m | J. Am. Chem. Soc. 2012, 134, 16734−16741

Journal of the American Chemical Society
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oligonucleotides containing 1 and 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
mgreenberg@jhu.edu
Notes
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The authors declare no competing financial interest.
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We are grateful for generous financial support from the
National Institute of General Medical Sciences (GM-063028).
We thank Professor Jack Norton and Mr. Will Kender for
helpful discussion with the kinetic analysis and the reviewers for
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