Reactivity of N3-Methyl-2′-Deoxyadenosine in Nucleosome Core Particles

Abstract

N3-Methyl-2′-deoxyadenosine (MdA) is the major dA methylation product in duplex DNA. MdA blocks DNA replication and undergoes depurination at significantly higher rates than the native nucleotide from which it is derived. Recent reports on the effects of the nucleosome core particle (NCP) environment on the reactivity of N7-methyl-2′-deoxyguanosine (MdG) inspired this investigation concerning the reactivity of MdA in NCPs. NCPs containing MdA at selected positions were produced using a strategy in which the minor groove binding Me-Lex molecule serves as a sequence specific methylating agent. Hydrolysis of the glycosidic bond in MdA to form abasic sites (AP) is suppressed in a NCP. Experiments using histone variants indicate that the proximal, highly basic N-terminal tails are partially responsible for the decreased depurination rate constant. MdA also forms cross-links with histone proteins. The levels of MdA-histone DNA–protein cross-links (DPC_MdA) decrease significantly over time and are replaced by those involving AP. The time dependent decrease in DPC_MdA is attributed to the reversibility of their formation and the relatively rapid rate of AP formation from MdA. Overall, MdA reactivity in NCPs qualitatively resembles that of MdG.

Introduction

DNA alkylation is a ubiquitous and biologically significant form of DNA damage.¹⁻³ Alkylated nucleotides can be mutagenic but are also a source of cytotoxicity induced by chemotherapeutic agents. Of the large variety of alkylating groups, methylation is the smallest and most common. Biologically important examples of methylating agents include current chemotherapeutic agents temozolomide (TMZ) and dacarbazine (DCZ) as well as N-methyl nitrosourea (MNU), dimethyl sulfate (DMS), and methylmethanesulfonate (MMS, Chart 1). N7-Methyl-2′-deoxyguanosine (MdG, 60–80%), N3-methyl-2′-deoxyadenosine (MdA, 10–20%), and O6-methyl-2′-deoxyguanosine (OmG, ≤5%) are the major DNA methylation products (Scheme 1). Although OmG is produced in the lowest chemical yield among the three products, the propensity of polymerases to misincorporate thymidine opposite it is believed to significantly contribute to the mutagenic and cytotoxic effects of methylating agents.² The correlation between cellular resistance to cytotoxic DNA methylating agents and levels of the OmG repair enzyme, O6-methylguanine-DNA methyltransferase, also attests to the biological significance of the lesion.¹ In contrast, other than being a source of abasic sites (AP), MdG was believed to have little biological consequence.^1,2 For instance, MdG was not believed to be cytotoxic or mutagenic.^4,5 However, recently, human DNA polymerase eta was found to bypass MdG with reduced fidelity.⁶ In addition, we determined that histone proteins form reversible DNA–protein cross-links (DPCs) with the lesion.^7,8 DPC_MdG formation could significantly enhance the biological consequences of MdG, and their discovery fortuitously coincides with exciting discoveries concerning DPC repair in general.⁹⁻¹³ The cellular lifetime of MdA is considerably shorter than that of MdG.¹⁴ Unlike MdG, MdA blocks DNA replication and is cytotoxic.¹⁵ MdA also undergoes depurination in free DNA more rapidly than does MdG. Furthermore, it has been shown that N3-methyladenine excision by base excision repair enzymes attenuates its toxicity.¹⁶ Therefore, any alteration in the persistence of MdA in NCPs may play an important role in its toxicity. Herein, we describe experiments using well-defined nucleosome core particles (NCPs) containing site-specific MdA to determine how the highly positively charged protein environment in chromatin affects the reactivity of this biologically significant DNA methylation product.

Chart 1. DNA Methylating Agents.

Scheme 1. Formation and Reactivity of Methylated Purines.

Alkylated purines undergo depurination >2000 times faster than the undamaged nucleotides.^3,17 The half-life of MdG in free DNA is 87–338 h.⁷ The rate constants for depurination (k_Hyd, Scheme 1) of MdG are suppressed as much as almost 6-fold in a NCP compared to that in free DNA. The slowest k_Hyd determined within the NCP was at MdG₁₂₃ (1 × 10^–7 s^–1, t_1/2 = 1930 h). The magnitude of the decrease in k_Hyd correlates with the proximity of the lesion to the highly positively charged N-terminal histone tails. MdA undergoes depurination in free DNA even more rapidly than MdG. In addition, like MdG, methylation creates an electrophilic species that could react with nucleophilic residues within histone proteins to form DPCs. Consideration of the MdA electronic structure suggests that DPC formation will occur from histone attack in the minor groove, presumably at the C2-position, whereas MdG likely reacts at the C8-position within the major groove of duplex DNA (Scheme 1).

Experimental Procedures

General Methods

Oligonucleotides used for polymerase chain reaction were purchased from Integrated DNA Technologies. Oligonucleotides for preparing 145-mer 601 DNA were synthesized on an Applied Biosystems Incorporated 394 oligonucleotide synthesizer. The oligonucleotide synthesis reagents were purchased from Glen Research (Sterling, VA). T4 polynucleotide kinase (PNK), terminal transferase, and proteinase K were from New England Biolabs. γ-³²P-ATP and α-³²P-cordycepin triphosphate were purchased from PerkinElmer. All experiments were performed in clear siliconized tubes from Bio Plas Incorporated. Quantification of radiolabeled oligonucleotides was done using a Molecular Dynamics Phosphorimager 840 equipped with ImageQuant TL software. 1-Methyl-4-(1-methyl-4-[3-(methoxysulfonyl)propanamido]pyrrole-2-carboxamido)pyrrole-2-carboxamido)propane (Me-Lex) was synthesized as previously described.¹⁸

Methylation of 601 DNA and NCPs by Me-Lex

Preparation of 601 DNA and NCPs with ³²P-labeling at the 5′-terminus of the top strand (1–145) or the 3′-terminus of the bottom strand (146–290) is described in the Supporting Information. Me-Lex (0.25 mg, 0.55 μmol) was dissolved in DMSO (55 μL) to a concentration of 10 mM, and then added to 5′- or 3′-³²P labeled free 601 DNA or NCPs (45 μL, 0.38 μM, ∼200,000 cpm) to a final concentration of 0.5 mM. The reaction mixture was incubated at 37 °C. An aliquot (15 μL) was taken after 0.5–1.5 h and stored on dry ice. All samples were phenol-chloroform extracted (equal volume), followed by ethanol precipitation (twice). The DNA was resuspended in 10 mM HEPES buffer (45 μL, pH 7.5) and heated at 90 °C for 15 min. The DNA was then ethanol precipitated, resuspended in piperidine solution (50 μL, 1 M), and heated (90 °C, 30 min). Piperidine was removed by evaporating to dryness in a speed vacuum. The products were resuspended in 95% formamide loading buffer (10 μL), and a portion of the sample (∼12,000 cpm) was analyzed by 10% denaturing PAGE. The gel (40 × 32 × 0.04 cm³) was run at room temperature under limiting power (50 W) until the bromophenol blue migrated to the bottom.

Generation of 601 DNA Containing Site-Specifically Incorporated MdA

The 145-mer 601 DNA (200 pmol) and the scaffold (427 pmol, Figure S2) were combined in a buffer containing 10 mM phosphate (pH 7.2) and 100 mM NaCl. The mixture (∼1.1 mL) was heated at 90 °C for 30 s, followed by slowly cooling to room temperature. The hybridized DNA was mixed with 55 μL of a 10 mM Me-Lex solution in DMSO and incubated at 37 °C for 2 h (MdA_58–59) or 4 h (MdA_234–235). The methylated DNA was concentrated, and the unreacted Me-Lex and reaction by-products were removed by nine rounds of buffer exchange with a 10 mM HEPES buffer (pH 7.5) using a 10 kDa Amicon centrifugal filter at 4 °C. The methylated DNA product (50 μL, ∼200 pmol) was stored at −80 °C until further use.

Characterization of 601 DNA Containing Site-Specifically Incorporated MdA

The 145-mer MdA containing DNA was ³²P-labeled at the 5′-terminus (MdA_58–59) or 3′-terminus (MdA_234–235). For 5′-³²P labeling, the reaction mixture (20 μL) containing DNA (20 pmol), 1× PNK buffer (70 mM Tris–HCl, pH 7.6, 10 mM MgCl₂, 5 mM DTT), γ-³²P ATP (30 μCi), and T4 PNK (30 units) was incubated at room temperature for 3 h. For 3′-³²P labeling, the reaction mixture (20 μL) containing DNA (20 pmol), 1× terminal transferase buffer (20 mM Tris–acetate, pH 7.9, 50 mM CH₃COOK, 10 mM Mg(CH₃COO)₂), CoCl₂ (0.25 mM), α-³²P-cordycepin triphosphate (30 μCi), and terminal transferase (60 units) was incubated at room temperature for 3 h. Free γ-³²P-ATP and α-³²P-cordycepin triphosphate were removed using Sephadex G-50 resin (1 mL). To determine the extent of methylation at nucleotide resolution (methylation efficiency), the labeled DNA (130 fmol, ∼100,000 cpm) was diluted into 10 mM HEPES (50 μL, pH 7.5), heated (90 °C, 15 min), and then ethanol precipitated. The DNA pellet was resuspended in piperidine (50 μL, 1 M) and heated (90 °C, 30 min). To determine the AP content, the labeled DNA (130 fmol, ∼100,000 cpm) was subjected to hot piperidine treatment (50 μL, 1 M, 90 °C, 30 min) without prior heating. Piperidine was removed by evaporating to dryness in a speed vacuum. The DNA was resuspended in 95% formamide (10 μL), and a portion of the sample (∼10,000 cpm) was analyzed by 10% denaturing PAGE. The gel (40 × 32 × 0.04 cm³) was run under limiting power (50 W) at room temperature until the xylene cyanol migrated to the bottom.

Reconstitution of NCPs Containing Site-Specifically Incorporated MdA

A mixture (10 μL) containing the complementary 145-mer (30 pmol), 10 mM phosphate buffer (pH 7.2), and 100 mM NaCl was heated at 90 °C for 1 min and immediately cooled on ice. The ³²P-labeled MdA containing DNA (20 μL, 20 pmol) was added and incubated at room temperature for 1 h. Salmon sperm DNA (20 μg, ∼168 pmol of 185-bp DNA) and the hybridized 145-bp MdA containing DNA (2 pmol, ∼4 million cpm) were combined in a siliconized tube to a final volume of 20 μL in the presence of 2 M NaCl and 0.1 mg/mL BSA. Histone octamer (1.2 equiv of total DNA) was added, and the mixture (22–30 μL) was incubated at 4 °C for 30 min before beginning a series of dilutions using the nucleosome reconstitution buffer (10 mM HEPES, pH 7.5, 1 mM EDTA, 0.1 mg/mL BSA) at 4 °C. Dilution #: volume of buffer added in μL, incubation time in minutes: 1: 24, 60; 2: 12, 60; 3: 12, 60; 4: 20, 30; 5: 20, 30; 6: 40, 30; 7: 100, 30; 8: 200, 30. After the final dilution (total volume ∼450 μL), any precipitate was pelleted via a brief spin (1 min, 13,000g) at 4 °C. A small aliquot (5 μL, ∼40,000 cpm) was removed, mixed with 3 μL of 40% sucrose, and analyzed by 6% native PAGE. The gel (10 × 8 × 0.15 cm³) was run at 4 °C under limiting power (3 W) until the bromophenol blue band migrated to the bottom.

Determining AP Yields from NCPs Containing Site-Specifically Incorporated MdA

NCPs (140 μL, ∼1 million cpm) containing site-specifically incorporated MdA were incubated at 37 °C. Aliquots (15 μL) were removed at appropriate times and stored at −80 °C until the final time point. To determine the amounts of AP, aliquots (10 μL) were treated with proteinase K (2.4 units) at room temperature for 30 min, followed by phenol–chloroform extraction (equal volume) and ethanol precipitation. The DNA was resuspended in piperidine (50 μL, 1 M) and heated (90 °C, 30 min). Piperidine was removed by evaporating to dryness in a speed vacuum. The samples were resuspended in 95% formamide loading buffer (10 μL), and a portion of the sample (∼20,000 cpm) was analyzed by 10% denaturing PAGE. The gel (40 × 32 × 0.04 cm³) was run at room temperature under limiting power (50 W) until the xylene cyanol migrated to the bottom. The normalized AP yield at each MdA (MdA_n) was calculated using eq 1. The rate constant for MdA hydrolysis (k_Hyd) was calculated by fitting the disappearance of MdA (1-normalized AP yield at MdA_n) to a first-order reaction.

1

Determining DPC Yields from MdA and AP in NCPs Containing Site-Specifically Incorporated MdA

NCPs (120 μL/∼850,000 cpm for 5.5 h of incubation, 25 μL/∼180,000 cpm for 48 h of incubation) containing MdA_58–59 or MdA_234–235 were incubated at 37 °C for 5.5 or 48 h, followed by 10% SDS PAGE analysis. The gel (20 × 16 × 0.1 cm³) was run at 4 °C under limiting power (3 W) until the bromophenol blue migrated to the bottom. The DPC bands were cut out from the gel, crushed, and eluted overnight at 4 °C in a buffer (10 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM EDTA) containing SDS (0.1%) and proteinase K (4 units). The gel particles were filtered using a polyprep column (BioRad). The filtrate was mixed with proteinase K (4 units) and incubated at room temperature for 10 min. Salmon sperm DNA (10 μg) was then added, followed by ethanol precipitation. The DNA samples (5000–10,000 cpm) were resuspended in H₂O (100 μL). To determine the total DPC yields (DPC_Total) from MdA and AP, the DNA samples (45 μL) were mixed with 100 mM HEPES buffer (5 μL, pH 7.5). The mixture was heated (90 °C, 15 min), followed by ethanol precipitation. The DNA samples were then subjected to hot piperidine treatment (1 M, 90 °C, 30 min). To determine the amount of DPC from AP (DPC_AP), an aliquot (45 μL) of the DNA samples isolated from the SDS PAGE was mixed with piperidine to a final concentration of 1 M and subjected to heating (90 °C, 30 min). Piperidine was removed by evaporating to dryness in a speed vacuum. DNA samples were resuspended in 95% formamide loading buffer (5 μL), and a portion of the sample (∼1000 cpm) was analyzed by 10% denaturing PAGE. The gel (40 × 32 × 0.04 cm³) was run at room temperature under limiting power (50 W) until the xylene cyanol migrated to the bottom. The yield of DPC from MdA (DPC_MdA) was calculated by subtracting the amount of DPC_AP from DPC_Total.

Results and Discussion

Preparation of Nucleosome Core Particles Containing MdA

Neither MdG nor MdA can be incorporated into DNA using solid phase synthesis due to their chemical instability. MdG was successfully incorporated at specific positions using DNA polymerase and the corresponding deoxyribonucleotide triphosphate.⁷ However, polymerase incorporation of MdA using the corresponding deoxyribonucleotide triphosphate was impractical due to the difficulty in synthesizing 3-methyl-2′-deoxyadenosine and its faster depurination than that of MdG.¹⁹ Random incorporation of MdA by treating NCPs with a methylating agent (e.g., methylmethanesulfonate) was also impractical because dG was the major modification site (Figure S3).

Consequently, we took advantage of the ability of the N-methylpyrrole carboxamide, Me-Lex, to selectively introduce MdA into DNA.^18,20,21 The Widom 601 sequence is a good one for reaction with Me-Lex because it contains multiple regions rich in dA–T base pairs that are favored binding sites for N-methylpyrrole carboxamides.²² In free DNA, Me-Lex preferentially alkylated dA₅₈ and dA₅₉ in one strand and dA₂₃₄ and dA₂₃₅ in the complementary strand (Figure 1 and Figure S4). These nucleotides are part of the 5′-d(T₅₆TAAAA₆₁) sequence. Two other dA/T rich sequences are alkylated to a lesser extent. Alkylation was biased toward dA₅₈ in the top strand when the NCP was reacted with Me-Lex (Figure S4).

Figure 1.

Me-Lex alkylation of free 601 DNA. The length of the lines is proportional to the amount of strand damage detected.

Detailed studies of MdA reactivity were carried out on NCPs in which the duplex region comprising 5′-d(T₅₆TAAAA₆₁) was alkylated. Selective methylation of this region was achieved by hybridizing one of the 145-mer strands with an oligonucleotide that hybridizes to this region (Scheme 2, Figures S5 and S6). The overall alkylation yield exceeded 60% at dA₅₈ and dA₅₉ and was ∼50% at dA₂₃₄ and dA₂₃₅ in the complementary strand. The ratio of MdA₅₉:MdA₅₈ or MdA₂₃₄:MdA₂₃₅ did not change as a function of Me-Lex concentration or reaction time (Figures S5 and S6), indicating that each strand was alkylated only once. The 145-mer DNA was then ³²P-labeled at the 5′-terminus for the strand containing MdA₅₈ and MdA₅₉ (top strand) or at the 3′-terminus for the strand containing MdA₂₃₄ and MdA₂₃₅ (bottom strand). The ³²P-labeled DNA was hybridized with its complement, and the NCPs were reconstituted using refolded octamers comprised of recombinantly purified Xenopus laevis histone proteins that were expressed in E. coli (Figures S9 and S10). Approximately 5% of MdA was converted to AP during the entire DNA and NCP preparation process. The correct positioning of the DNA wrapping on octamer was confirmed by DNase I digestion (Figures S9 and S10). NCPs with a reconstitution efficiency larger than 93% were used for the experiments.

Scheme 2. Selective Introduction of MdA into DNA Using Me-Lex.

N3-Methyl-2′-deoxyadenosine Depurination

The rate constants for hydrolysis (k_Hyd, Scheme 1) were determined by measuring the amount of AP present as a function of time and normalizing for the MdA level at a particular position (Tables 1–4). AP levels were determined by taking advantage of the observation that hot piperidine treatment did not cleave DNA at MdA (Scheme 3, Figure S7). Consequently, direct hot piperidine treatment of DNA following proteinase K digestion selectively cleaves the DNA at AP sites, facilitating their quantitation following denaturing PAGE analysis, whereas the same process following heating yields strand scission at MdA and AP sites. Of the four positions at which depurination was examined, k_Hyd in free DNA ranged between 8 × 10^–6 and 10.7 × 10^–6 s^–1 at three sites. MdA₅₉ depurinates approximately 2–2.5 times faster than the other positions in free DNA. Hydrolysis of MdA was suppressed at all four positions within wild type NCPs. However, MdA₅₉, the most reactive alkylation site in free DNA, was affected the least within NCPs. The half-life for MdA₅₉ depurination in the NCP was 1.9-fold longer than that in free DNA. In contrast, the relative half-lives (rel. t_1/2 = t_1/2(NCP)/t_1/2(free DNA)) for MdA depurination at the other three positions ranged from 2.9 to 3.6.

Table 1. MdA₅₈ Depurination Kinetics as a Function of Environment.

substrate	kHyd (×10–6 s–1)	t1/2 (h)	rel. t1/2c
free DNAb	8.0 ± 1.1	24.4 ± 3.3
WTb	2.2 ± 0.1	86.7 ± 3.2	3.6 ± 0.1
H3 Del.b	2.3 ± 0.5	84.7 ± 17.2	3.5 ± 0.9
H4 Del.b	2.9 ± 0.1	67.3 ± 0.1	2.8 ± 0.4
H3, H4 Del.a	3.1 ± 0.3	61.9 ± 6.8	2.5 ± 0.4

^aData are the average ± std. dev. of three replicates.

^bData are the average ± std. dev. of two experiments each consisting of three replicates.

^cRel. t_1/2 = t_1/2(NCP)/t_1/2(free DNA).

Table 4. MdA₂₃₅ Depurination Kinetics as a Function of Environment.

substratea	kHyd (×10–6 s–1)	t1/2 (h)	rel. t1/2b
free DNA	10.7 ± 2.6	18.8 ± 4.9
WT	3.0 ± 0.1	53.9 ± 2.5	2.9 ± 0.8
H3 Del.	4.8 ± 0.8	41.0 ± 7.0	2.2 ± 0.7
H4 Del.	6.2 ± 0.4	31.1 ± 2.1	1.6 ± 0.4
H3, H4 Del.	6.5 ± 1.1	29.1 ± 5.1	1.5 ± 0.5

^aData are the average ± std. dev. of three replicates.

^bRel. t_1/2 = t_1/2(NCP)/t_1/2(free DNA).

Scheme 3. Distinguishing MdA from AP.

Table 2. MdA₅₉ Depurination Kinetics as a Function of Environment.

substratea	kHyd (×10–6 s–1)	t1/2 (h)	rel. t1/2b
free DNA	20.4 ± 1.3	9.5 ± 0.6
WT	10.7 ± 0.3	18.0 ± 0.5	1.9 ± 0.1
H3 Del.	10.8 ± 1.2	18.0 ± 1.9	1.9 ± 0.2
H4 Del.	13.0 ± 0.8	14.8 ± 1.0	1.6 ± 0.1
H3, H4 Del.	13.4 ± 2.8	14.4 ± 2.4	1.5 ± 0.3

^aData are the average ± std. dev. of three replicates.

^bRel. t_1/2 = t_1/2(NCP)/t_1/2(free DNA).

The NCP effect on MdA depurination at these three positions is very similar to the effect on MdG₈₉ (rel. t_1/2 = 2.9) but significantly smaller than that on MdG₁₂₃ (rel. t_1/2 = 5.7).⁷ Experiments with histone variants revealed that the proximal histone H2B N-terminal tail contributes significantly to the suppression of k_Hyd at MdG₁₂₃. MdG₈₉ and the MdA positions examined here are proximal to the histone H3 and H4 N-terminal tails (Figure 2). A molecular model based upon superimposed X-ray crystal structures of NCPs (pdb: 1kx5 and 3lz0) indicates that this histone H4 tail can interact with the minor groove in the vicinity of all four MdA molecules.^23,24 This flexible portion of the protein also appears to be able to interact with the major groove in which MdA₂₃₄ and MdA₂₃₅ are located. In contrast, the histone H3 tail, which protrudes from the core between the two DNA gyres in the NCP must traverse the DNA backbone to access the minor groove containing the methylated dA’s. The major groove containing MdA₅₈ and MdA₅₉ is also relatively inaccessible to the histone H3 tail. However, the major groove in which MdA₂₃₄ and MdA₂₃₅ are positioned is in closer juxtaposition to the histone H3 tail.

Figure 2.

Nucleosome core particle structure in the vicinity of MdA₅₈, MdA₅₉, MdA₂₃₄, and MdA₂₃₅. (A) Major region damaged by Me-Lex showing histone H3 (blue) and H4 (green) N-terminal tails. (B) Zoomed in region showing C2-positions (red arrows) of alkylated dAs. The structure is generated by superimposing two NCP structures (pdb: 1kx5 and 3lz0).

These qualitative interpretations are largely borne out in kinetic studies involving NCPs containing histone variants. Deleting the 42 N-terminal amino acids from histone H3 has little if any effect on k_Hyd of MdA₅₈, MdA₅₉, or MdA₂₃₄ (Tables 1–3). However, rel. t_1/2 of MdA₂₃₅ is reduced from 2.9 to 2.2 when the histone H3 N-terminal tail is deleted. Deleting the histone H4 N-terminal tail increases k_Hyd at every MdA position. At one position, MdA₅₈, there is a small synergistic effect from deleting the N-terminal tails of histones H3 and H4 (Table 1).

Table 3. MdA₂₃₄ Depurination Kinetics as a Function of Environment.

substratea	kHyd (×10–6 s–1)	t1/2 (h)	rel. t1/2b
free DNA	8.5 ± 0.4	22.6 ± 1.1
WT	2.8 ± 0.2	68.7 ± 4.9	3.0 ± 0.3
H3 Del.	3.0 ± 0.7	66.2 ± 15.6	2.9 ± 0.7
H4 Del.	4.1 ± 0.2	47.5 ± 2.6	2.1 ± 0.2
H3, H4 Del.	3.8 ± 0.1	50.6 ± 1.3	2.2 ± 0.1

^aData are the average ± std. dev. of three replicates.

^bRel. t_1/2 = t_1/2(NCP)/t_1/2(free DNA).

Overall, the hydrolysis of MdA is similar to that of MdG in that it is significantly inhibited in a NCP. In addition, the proximal histone tail(s) has a similar effect on MdA depurination to that on the same reaction by MdG. It is also notable that the tails are not solely responsible for suppressing MdA hydrolysis in the NCPs relative to free DNA. This too was observed in experiments on MdG.

DNA–Protein Cross-Links at Positions Where N3-Methyl-2′-deoxyadenosine Is Incorporated

Inspired by the reversible DPC formation between MdG and histone proteins in NCPs, we examined whether MdA formed comparable products.⁷ Detecting DPCs by SDS PAGE is straightforward. Determination of their origin is more complicated because the AP sites formed upon depurination form transient DPCs.^8,25,26 DPCs between histones and AP (DPC_AP) or MdA (DPC_MdA) are distinguishable by taking advantage of the facile cleavage of the DNA in the former, whereas the DNA in DPC_MdA is uncleaved when subjected to alkaline conditions (Scheme 4). In practice, the DPCs are isolated via SDS PAGE and the proteins are enzymatically degraded. The DNA is then subjected to alkaline conditions and analyzed by denaturing PAGE. Uncleaved and cleaved DNA are attributed to DPC_MdA and DPC_AP, respectively.

Scheme 4. Distinguishing DPCs Formed from MdA and AP.

The DPC_Total (based upon the levels of MdA) at the respective alkylated positions ranged from approximately 3 to 5% after incubating at 37 °C for 5.5 h but increased to as much as ∼22% upon incubation for 48 h (Table 5). The DPC yields are considerably higher than what was observed in NCPs containing MdG.⁷ DPCs were not detected when the NCPs containing MdA were comprised of histone H4 tail deletion variants, indicating that this protein is solely responsible for cross-link formation (Figure S13). In addition, the fraction of DPCs resulting from reaction with MdA (DPC_MdA) decreased at all positions over time (Figure 3). For instance, after incubating NCPs containing MdA₅₈ and MdA₅₉ for 5.5 h, DPC_MdA accounted for ∼42 and ∼28% of the DPCs, respectively. However, the contribution of DPC_AP in these NCPs to DPCs rose to more than 92% after 48 h (Figure 3A,B). The contribution of DPC_MdA to DPC formation was even smaller when the alkylated purines were present in the complementary strand (Figure 3C,D). After 48 h, more than 97% of the DPCs originating from methylation of dA₂₃₄ and dA₂₃₅ were attributable to AP lesions at these positions.

Table 5. DNA–Protein Cross-Link Yields as a Function of Incubation Time.

	DPCTotal yielda (%)
position	5.5 h	48 h
MdA58	3.2 ± 0.3	17.8 ± 1.2
MdA59	3.7 ± 0.4	22.2 ± 2.5
MdA234	4.8 ± 0.7	19.5 ± 2.2
MdA235	4.4 ± 0.6	17.2 ± 4.5

^aData are the average ± std. dev. of three replicates.

Figure 3.

Distribution of DPCs as a function of time from (A) MdA₅₈, (B) MdA₅₉, (C) MdA₂₃₄, and (D) MdA₂₃₅.

In general, the fraction of DPC_MdA is considerably smaller at all times than that of DPC_MdG.⁷ This could be attributed to the irreversible nature of the depurination reaction and the significantly greater k_Hyd for MdA. Although DPC formation from AP, MdA, and MdG is reversible, depurination eliminates the possibility that the methylated nucleotide can contribute to this product family. Hence, the higher rate constants for MdA depurination reduce its contribution to DPC_MdA compared to the longer lived MdG. It is also possible that the C2-position of MdA, the presumed site of nucleophilic attack, which lies in the relatively narrow minor groove, may be less accessible to the histone tail(s). It is not clear from these experiments why MdA₅₈ and MdA₅₉ form DPC_MdA more readily than the methylated nucleotides on the complementary strand. There is no correlation between k_Hyd and DPC_MdA yields. For instance, the rate constants for depurination of MdA₂₃₄ and MdA₂₃₅ are very similar to that of MdA₅₈, yet DPC_MdA accounts for a much higher fraction of DPCs at the latter site. The absence of a correlation between k_Hyd and DPC yield is also evident at position 59. Hydrolysis at MdA₅₉ is faster than that at the other three examined positions. However, it produces higher yields of DPC_MdA than do MdA₂₃₄ and MdA₂₃₅. In addition, inspection of the NCP X-ray structure (Figure 2) does not suggest that the C2-positions of MdA₅₈ and MdA₅₉ are more accessible to the N-terminal histone H4 tail than MdA₂₃₄ and MdA₂₃₅. One possibility is that the reversion rate constant for DPC_MdA involving MdA₂₃₄ and MdA₂₃₅ may be greater. However, we were unable to probe this possibility due to the heterogeneity of the DPC_total and low yields of DPC_MdA.

Summary

N3-Methyl-2′-deoxyadenosine is recognized to be a block to DNA replication and a source of abasic sites. Recent studies on the reactivity of closely related N7-methyl-2′-deoxyguanosine in nucleosome core particles prompted this investigation.⁷ MdA reactivity in NCPs parallels that of MdG. Specifically, the rate constant for MdA depurination is smaller in NCPs than in free DNA. The relative reduction is comparable to that observed for MdG in the same general region of the NCP. The proximal N-terminal histone H4 tail is a significant contributor to the observed suppression of depurination. However, the highly positively charged octameric core also plays a role in decreasing the rate constants for hydrolysis. MdA yields considerable amounts of DNA–protein cross-links. Although they are produced reversibly, this is to our knowledge the first evidence for the formation of DPCs between MdA and histone proteins. Unlike MdG, the vast majority of DPCs generated from MdA are ultimately between AP and the histone protein and not the alkylated nucleotide. The source of this difference is uncertain. Reversible formation of DPC_MdA and the more rapid depurination (k_Hyd) from MdA versus MdG may be one reason for the difference. In addition, the presumed need for nucleophilic attack of MdA by the histone tail(s) in the minor groove may also be a reason for the reduced levels of DPCs between MdA and histones compared to those involving MdG.

It is difficult to evaluate the role of decreased depurination of MdA in NCPs, as both MdA and AP are cytotoxic. DPCs are extremely deleterious because they block DNA replication and transcription. DPCs between histone proteins and MdG have been detected in cells treated with methylmethanesulfonate.⁷ If DPC formation between histones and MdA in NCPs occurs in cells, they would also contribute to the cytotoxic effect of MdA even if formed in small amounts.

Glossary

Abbreviations

Me-Lex

1-methyl-4-(1-methyl-4-[3-(methoxysulfonyl)propanamido]pyrrole-2-carboxamido)pyrrole-2-carboxamido)propane

MdA

N3-methyl-2′-deoxyadenosine

MdG

N7-methyl-2′-deoxyguanosine

OmG

O6-methyl-2′-deoxyguanosine

DPC

DNA–protein cross-link

DPC_MdG

DPC between MdG and histone protein

DPC_MdA

DPC between MdA and histone protein

DPC_AP

DPC between AP and histone protein

DPC_Total

total DPCs

AP

abasic site

NCP

nucleosome core particle

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.9b00299.

• Experimental methods for the 601 DNA preparation, NCP characterization, and sample autoradiograms (PDF)

We are grateful for financial support from the National Institute of General Medical Sciences (GM-131736) to M.M.G. and from UNCW (Charles L. Cahill and CSURF Awards) to S.V.

The authors declare no competing financial interest.