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Cite This: Chem. Res. Toxicol. 2019, 32, 840−849

Epigenetic DNA Modification N6‑Methyladenine Inhibits DNA Replication by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1 Bianbian Li,†,‡ Ke Du,‡ Shiling Gu,‡ Jiayu Xie,‡ Tingting Liang,‡ Zhongyan Xu,‡ Hui Gao,† Yihui Ling,§ Shuguang Lu,∥ Zhen Sun,*,† and Huidong Zhang*,‡ †

School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China Key Laboratory of Environment and Female Reproductive Health, West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, China § Institute for Chemical Carcinogenesis, Guangzhou Medical University, Xinzao, Panyu District, Guangzhou, China ∥ Department of Microbiology, College of Basic Medical Science, Third Military Medical University, Chongqing, China

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‡

ABSTRACT: N6-methyladenine (6mA), a newly identified epigenetic modification, plays important roles in regulation of various biological processes. However, the effect of 6mA on DNA replication has been little addressed. In this work, we investigated how 6mA affected DNA replication by DNA polymerase of Pseudomonas aeruginosa Phage PaP1 (gp90 exo−). The presence of 6mA, as well as its intermediate hypoxanthine (Hyp), inhibited DNA replication by gp90 exo−. The 6mA reduced dTTP incorporation efficiency by 10-fold and inhibited next-base extension efficiency by 100-fold. Differently, dCTP was preferentially incorporated opposite Hyp among four dNTPs. Gp90 exo− reduced the extension priority beyond the 6mA:T pair rather than the 6mA:C mispair and preferred to extend beyond Hyp:C rather than the Hyp:T pair. Incorporation of dTTP opposite 6mA and dCTP opposite Hyp showed fast burst phases. The burst rate and burst amplitude were both reduced for 6mA compared with unmodified A. Moreover, the total incorporation efficiency (kpol/Kd,dNTP) was decreased for dTTP incorporation opposite 6mA and dCTP incorporation opposite Hyp compared with dTTP incorporation opposite A. 6mA reduced the incorporation rate (kpol), and Hyp increased the dissociation constant (Kd,dNTP). However, 6mA or Hyp on template did not affect the binding of DNA polymerase to DNA in binary or ternary complexes. This work provides new insight into the inhibited effects of epigenetic modification of 6mA on DNA replication in PaP1.

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hydrolysis.3 Genomic DNA can also be methylated by various endogenous methyltransferases to generate N4-methylcytosine (4mC), 5-methylcytosine (5mC), or N6-methyladenine (6mA).4,5 They are considered as signaling or epigenetic modifications since they were predicted not to disrupt base pairing.6−8 Differently, N1-methyladenine (1mA) and N3methylcytosine (3mC) are considered as DNA lesions because these modifications disrupt H-bond formation.9

INTRODUCTION Efficient and accurate DNA replication is very important in keeping genomic integrity during cell division and proliferation. However, various exogenous and endogenous factors produce diverse DNA lesions or modifications, which damage DNA replication machinery.1,2 These modifications or lesions may increase misincorporation frequency, produce frameshift deletion, or block DNA replication, possibly altering oncogenes and tumor suppressors and further inducing tumor and cancer.3 DNA lesions are produced through various chemical reactions, such as oxidation, alkylation (which may involve cross-linking), deamination, photoaddition, coordination, and © 2019 American Chemical Society

Special Issue: Epigenetics in Toxicology Received: November 14, 2018 Published: April 2, 2019 840

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Chemical Research in Toxicology

Figure 1. Structure illustration of adenine (A), N6-methyladenine (6mA), and hypoxanthine (Hyp), and a scheme of their conversions.

work, we will investigate how gp90 exo− bypasses 6mA and its intermediate hypoxanthine (Hyp). Our results show that 6mA and Hyp partially inhibited primer extension and reduced the efficiencies of correct dNTP incorporation, next-base extension, and burst incorporation, providing new insight in the inhibited effects of epigenetically modified 6mA on DNA replication in P. aeruginosa phage PaP1.

6mA is primarily present in prokaryotes.10 Recently, 6mA was also found in eukaryote genomes,11 including green algae,12 nematodes,13 fungi,14 insects,15 vertebrates,16 mouse embryonic stem cells,17 Arabidopsis thaliana,18 rice,19 zebrafish and pig,4 and human.20 The genomic distribution of 6mA varies among different species. 6mA levels are relatively high in unicellular eukaryotes but very low in metazoans.21 About 70% of 6mA sites are located in genes and concentrated around the transcription start sites.22 6mA is generated by methylation of adenine (A) catalyzed by MT-A70 family methylases in eukaryotes23,24 (Figure 1). 6mA can be oxidized by AlkB family enzymes to form 6hydroxymethyladenine (6hmA), which releases its formaldehyde group to produce A.24,25 Alternatively, 6mA can also be deaminated to generate hypoxanthine (Hyp, Figure 1), which is subsequently removed by base excision repair via AlkA family enzymes, and then the correct dATP is incorporated before DNA ligation.24,26 Adenine can also be converted to Hyp via oxidative deamination during inflammation and oxidation by nitric oxide.27,28 In C. elegans, DNA methyltransferase (DAMT-1) and demethylase (NMAD-1) regulate 6mA levels and crosstalk between methylations of adenines.13 In prokaryotes, 6mA is a signaling or epigenetic mark and distinguishes the self from foreign DNA.6 In bacteria, 6mA plays important roles in regulation of DNA mismatch repair, chromosome replication, cell cycle regulation, transcription, and restriction−modification.12 In eukaryotes, 6mA was also found as a heritable epigenetic modification.13 During early embryo development in metazoans, the elevated levels of 6mA serve as an important epigenetic mark to control transcription.21 In green alga Chlamydomonas, 6mA accumulation is related to gene activation.12 In vitro, 6mA hinders DNA synthesis on a DNA or RNA template by a large fragment of Bst DNA polymerase.29 Recently, 6mA was also found in the genome of the Pseudomonas aeruginosa phage (PaP1).30 The PaP1 genome contains 91 715 base pairs and 51 6mA’s. However, whether and how 6mA affects DNA replication in PaP1 is unknown. Recently, we have identified that the DNA polymerase of PaP1, gp90, is an A-family processive DNA polymerase containing 3′ → 5′ exonuclease activities on ssDNA and dsDNA.31 Exonuclease-deficient gp90 exo− can bypass 8-oxoG error-free,32 was partially inhibited by an alkylation lesion, O6MeG,33 and was completely blocked by an abasic site.1 In this

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MATERIALS AND METHODS

DNA Substrates and Proteins. [γ-32P] ATP was purchased from PerkinElmer Life Sciences (Boston, MA). Oligonucleotides were synthesized by Midland Certified Reagent Co. (Midland, TX). T4 polynucleotide kinase and dNTPs were from Amersham Biosciences (Piscataway, NJ). Gp90 exo− was purified as described previously.1 Other reagents are of the highest quality commercially available. Prime Extension by gp90 exo− in the Presence of Four dNTPs. A 35-mer template containing A, 6mA, or Hyp was annealed to a 32P-labeled 24-mer primer (Table 1). Primer extension was

Table 1. Oligodeoxynucleotides 24-mer 25T-mer 25C-mer 27-mer 35-mer a

5′-TCGCATAGATCTCAGGTCAAGTAC-3′ 5′-TCGCATAGATCTCAGGTCAAGTACT-3′ 5′-TCGCATAGATCTCAGGTCAAGTACC-3′ 5′-biotin-TTTTCGCATAGATCTCAGGTCAAGTACdd-3′ 3′-AGCGTATCTAGAGTCCAGTTCATGA*TCGCTTACGA5′a

A*: A, 6mA, or Hyp.

performed by mixing 20 nM DNA substrates and varying concentrations of gp90 exo− with 350 μM each of four dNTPs at 37 °C for 1 min in buffer A (pH 7.5), which contains 30 mM Mg2+, 40 mM Tris-HCl (pH 7.5), 50 mM potassium glutamate, and 10 mM DTT. Reactions were quenched by the addition of 95% formamide (v/v), xylene cyanol, bromphenol blue, and 20 mM EDTA. Products were separated on a 20% polyacrylamide (w/v)/7 M urea gel, visualized using a phosphor imaging screen, and quantified with Quantity One software.33,34 Steady-State Kinetic Analysis of Single-Nucleotide Incorporation and Next-Base Extension. Reactions were performed using a 32P-labeled 24-mer or 25-mer primer/35-mer template containing A, 6mA, or Hyp (Table 1) with a molar ratio of gp90 exo− to DNA substrate <10% in buffer A at 37 °C.26 The extent of primer extension was controlled <0.20 by adjusting polymerase concentrations and reaction time.26 Reactions were quenched, and products were analyzed and quantified. kcat and Km values were obtained by fitting using GraphPad Prism Version 6.0 (San Diego, CA). The misincorporation frequencies were obtained by dividing the 841

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Figure 2. Full-length primer extension beyond A, 6mA, or Hyp by gp90 exo−. Extension assays were performed by mixing 0, 2, 5, 10, or 20 nM gp90 exo−; 20 nM 32P-labeled 24-mer/35-mer DNA substrate containing A, 6mA, or Hyp; and 350 μM each of four dNTPs in buffer A at 37 °C for 1 min. The arrows depict the location of the substrate and 35-mer product. Percentages on top depict the conversions of primer to 35-mer product. Representative data from multiple experiments are shown.

Table 2. Steady-State Kinetic Parameters of Single-Nucleotide Incorporation Opposite A, 6mA, or Hyp by gp90 exo−a primer-template 5′-C 3′-GATC

5′-C 3′-G6mATC

5′-C 3′-GHypTC

dNTP dTTP dATP dGTP dCTP dTTP dATP dGTP dCTP dTTP dATP dGTP dCTP

kcat, ×10−3 s−1 27 4.2 1.7 2.5 5.9 3.7 2.0 1.1 3.4 3.5 0.8 3.6

± ± ± ± ± ± ± ± ± ± ± ±

c

0.01 0.04 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01

kcat/Km, μM−1 s−1

Km, dNTP, μM −3

(1.2 ± 0.1) × 10 6.8 ± 1.3 10 ± 1.5 2.8 ± 0.3 (2.8 ± 0.4) × 10−3 2.4 ± 0.1 14 ± 2.3 9.9 ± 1.4 0.44 ± 0.02 0.56 ± 0.07 0.47 ± 0.07 (2.4 ± 0.2) × 10−4

6.2 1.7 8.9 1.5 1.4 1.2 7.8 6.3 1.7

23 × 10−4 × 10−4 × 10−4 2.1 × 10−3 × 10−4 × 10−4 × 10−3 × 10−3 × 10−3 15

misincorporation frequencyb 2.7 × 10−5 7.4 × 10−6 3.9 × 10−5 7.1 6.7 5.7 5.2 4.2 1.1

× × × × × ×

10−4 10−5 10−5 10−4 10−4 10−4

efficiency relative to A:dTTP 1 3.7 × 104-fold 1.4 × 105-fold 2.6 × 104-fold 11-fold less 1.5 × 104-fold 1.6 × 105-fold 1.9 × 105-fold 2.9 × 103-fold 3.7 × 103-fold 1.4 × 104-fold 1.5-fold less

less less less less less less less less less

a

The extent of incorporation was controlled <20% by adjusting polymerase concentration and reaction time. bMisincorporation frequency is defined as (kcat/Km)incorrect‑dNTP/(kcat/Km)correct‑dTTP‑or‑dCTP. cThe standard errors were derived using Prism. Biacore T200 (Uppsala, Sweden) as described previously.37,38 A 27mer primer containing a Cdd (double deoxycytosine) at its 3′ terminus and a biotin at its 5′ terminus was annealed to a 35-mer template containing A, 6mA, or Hyp (Table 1). DNA was immobilized on an SA chip (600 RU). Gp90 exo− (10−800 nM) was flowed over the SA chip in buffer B (10 mM DTT, 30 mM Mg2+, 50 mM potassium glutamate, and 40 mM Tris-HCl (pH 7.5)) for 120 s. Binding signals at 120 s versus gp90 exo− concentrations were fitted to eq 4.

incorporation efficiency (kcat/Km) of each misincorporated dNTP by that of dCTP.35,36 The standard errors were derived using Prism. Presteady-State Kinetic Analysis of Nucleotide Incorporation. Presteady-state kinetic assays were performed as described previously1 with minor changes. Reactions were performed by rapidly mixing 80 nM gp90 exo− and 120 nM 32P-labeled 24-mer/35-mer DNA mixture with an equal volume of 1 mM dNTP in buffer A at 37 °C. The product concentrations and time were fitted to eq 1, corresponding to dNTP incorporation in the burst phase and in the steady-state phase. All parameters and standard errors were derived using Prism. y = A(1 − ek pt ) + ksst

Y = B × RU /(B + Kd)

where Y is binding response signal at 120 s, RU; RU is the binding signal at 120 s, RU; B is gp90 exo− concentration, nM; and Kd is the approximate dissociation constant, nM. The binding was also determined in the presence of additional 1 mM dTTP or dCTP. All experiments were carried out thrice, and standard errors were derived using Prism.

(1)

where y is the product concentration, nM; A is the burst amplitude, nM; kp is the burst rate, s−1; t is the time, s; and kss is the steady-state phase rate, nM s−1. The maximal burst rate (kpol) and dNTP equilibrium dissociation constant (Kd,dNTP) were determined in reactions with 100 nM DNA and 200 nM gp90 exo− at different dNTP concentrations. The product concentrations and time were fitted to eq 2 to obtain each burst rate (kobs). Then, the burst rates and dNTP concentrations were fitted to eq 3 to estimate kpol and Kd,dNTP values. y = A(1 − e−kobst )

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RESULTS Full-Length Primer Extension by gp90 exo−. The effects of 6mA on full-length primer extension by gp90 exo− were investigated. Gp90 exo− could extend the 24-mer primer to a 35-mer product on an unmodified template, while this extension was relatively inhibited by 6mA and Hyp (Figure 2). The percentages on the top of each gel show the conversion of a primer to a 35-mer product. No intermediates were observed, indicating that this DNA polymerase still showed high processivity in the bypass of 6mA or Hyp. These results showed that 6mA, as well as its intermediate Hyp, partially inhibited DNA replication by gp90 exo−.

(2)

kobs = k pol[dNTP]/([dNTP] + Kd,dNTP)

(4)

(3)

−1

where kobs is burst incorporation rate, s ; kpol is the maximal burst rate, s−1; and Kd,dNTP is the dNTP equilibrium dissociation constant, μM. Surface Plasmon Resonance (SPR) Analysis of the Binding of gp90 exo− to DNA. Biophysical binding of gp90 exo− to the primer/template containing A, 6mA, or Hyp was determined using a 842

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Chemical Research in Toxicology Table 3. Steady-State Kinetic Parameters for Next-Base Extension beyond A, 6mA, or Hyp by gp90 exo−a primer-template 5′-CX 3′-GATC 5′-CX 3′-G6mATC 5′-CX 3′-GHypTC

primer X T C T C T C

kcat, ×10−3 s−1 2.2 0.80 4.1 1.7 1.1 1.3

± ± ± ± ± ±

0.08 0.02 0.04 0.01 0.05 0.05

Km,dATP, μM

kcat/Km, ×10−3 μM−1 s−1

efficiency relative to A:T pair

± ± ± ± ± ±

12 9.2 × 10−3 0.12 8.0 × 10−3 0.01 13

1 1300-fold less 100-fold less 1500-fold less 1200-fold less 0.90-fold less

0.20 87 34 220 110 0.10

0.05 11 7 35 15 0.03

a

The extent of extension was controlled <20% by adjusting enzyme concentration and reaction time. The standard errors were derived using Prism.

Figure 3. Presteady-state kinetic analysis of single-nucleotide incorporation by gp90 exo−. Gp90 exo− (80 nM) incubated with 120 nM 32P-labeled 24-mer/35-mer primer/template containing A (A), 6mA (B), or Hyp (C) was rapidly mixed with 1 mM of each individual dNTP in buffer A. Product concentrations versus time were fitted to eq 1 to obtain burst amplitude and burst rate kp. Representative data from multiple experiments are shown. Standard errors were derived using Prism.

Steady-State Kinetic Analysis of Nucleotide Incorporation Opposite A, 6mA, or Hyp. Km and kcat values were measured for dNTP incorporation opposite A, 6mA, or Hyp by gp90 exo− (Table 2). dTTP was preferentially incorporated opposite A. The misincorporation frequencies of three incorrect dNTPs were at the level of 10−5 to 10−6. The kcat values of the three incorrect dNTPs were decreased 6−16-fold relative to that of dTTP, while their Km values were 1000-fold higher than that of dTTP. Opposite 6mA, dTTP was preferentially incorporated. The misincorporation frequencies of three incorrect dNTPs were 10−4 to 10−5. The efficiency of dTTP incorporation opposite 6mA was 11-fold lower than that opposite A. Differently, dCTP was preferentially incorporated opposite Hyp, 4 orders of magnitude more efficiently than the other three dNTPs. The 5′ next nucleotide was T, precluding the possibility that dCTP was directly incorporated opposite the next nucleotide T through a −1 frameshift deletion. Taken together, 6mA on the template reduced dTTP incorporation efficiency, and Hyp on the template preferred dCTP incorporation. Steady-State Kinetic Analysis of Next-Base Extension. Km and kcat values of next-base extension beyond A, 6mA, or Hyp were measured (Table 3). T or C at the primer terminus was paired or mispaired with template A, 6mA, or Hyp, respectively. For template A, dATP incorporation opposite the 5′ next base T was about 1300-fold more efficient beyond the A:T pair (primer:template) than the A:C mispair, due to similar kcat values but a significantly decreased Km value. For template 6mA, dATP incorporation opposite the next base T was 100-fold less efficient beyond 6mA:T than beyond the A:T pair, indicating that 6mA inhibited the next-base extension. Additionally, the extension efficiency was only 15-fold preferential in extension beyond the 6mA:T pair rather than

the 6mA:C mispair, showing that 6mA greatly reduced the priority in extension beyond the correct pair rather than the mispair. The extension efficiency was about 1300-fold higher beyond Hyp:C than the Hyp:T pair. Therefore, gp90 exo− preferentially extended beyond the 6mA:T pair or Hyp:C pair but inhibited the extension efficiency and lost priority beyond the 6mA:T pair and was completely preferred to extension beyond Hyp:C rather than the Hyp:T pair. Presteady-State Kinetic Analysis. If DNA is a molar excessive relative to DNA polymerase, correct dNTP incorporation generally shows a burst phase and steady-state phase for most DNA polymerases.33 This biphasic feature demonstrates that the dissociation of the polymerase from DNA during the steady-state phase is much lower than dNTP incorporation during the burst phase.32,33 To determine the burst kinetic parameters of dNTP incorporation opposite A, 6mA, or Hyp by gp90 exo−, a molar excess of DNA compared to gp90 exo− was used. dTTP among four dNTPs was preferentially incorporated opposite A or 6mA and showed a biphasic feature with a burst rate kp of 14 s−1 or 0.81 s−1, respectively (Figure 3A,B). The incorporation of three incorrect dNTPs exhibited linear phases. Incorporation of dCTP opposite Hyp was preferential relative to the other three dNTPs and showed a biphasic phase with a kp value of 10 s−1 (Figure 3C). Incorporations of the other three dNTPs opposite Hyp showed linear phases. The 5′ next template nucleotide was T, excluding the possibility that dCTP was directly incorporated opposite the T via a −1 frameshift deletion. The burst rate of dTTP incorporation opposite A was 18-fold higher than that of dTTP incorporation opposite 6mA and 1.4-fold higher than that of dCTP incorporation opposite Hyp. The burst amplitude indicates the amount of productive gp90 exo−−DNA−dNTP ternary complex that could actively 843

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Figure 4. Presteady-state kinetic analysis of single dNTP incorporation opposite A, 6mA, or Hyp by gp90 exo−. Gp90 exo− (200 nM) incubated with 100 nM 32P-labeled 24-mer/35-mer primer/template was rapidly mixed with varying concentrations of dNTP to initiate reactions. Product concentrations versus time were fitted to eq 2 to obtain kobs at each dNTP concentration. Burst rates (kobs) versus dNTP concentrations were fitted to eq 3 to obtain kpol and Kd,dNTP values. (A, D) dTTP incorporation opposite A. (B, E) dTTP incorporation opposite 6mA. (C, F) dCTP incorporation opposite Hyp. Representative data from multiple experiments are shown. Standard errors were derived using Prism.

hardly impact the binding of gp90 exo− to DNA (Table 4, Figure 5).

catalyze dNTP incorporation. The burst amplitudes were estimated to 20 ± 1, 12 ± 0.8, and 36 ± 2 nM for dTTP incorporation opposite A, dTTP opposite 6mA, and dCTP opposite Hyp, respectively (Figure 3). 6mA reduced the burst amplitude and the amount of productive ternary complex. Then, kpol (the maximal burst rate) and Kd,dNTP (the dissociation constant of dNTP) were estimated by analysis of the burst rates as a function of dNTP concentrations. dTTP incorporation opposite A gave a kpol value of 16 ± 1 s−1 and a Kd,dTTP of 7.3 ± 0.5 μM (Figure 4A, D). For dTTP incorporation opposite 6mA, kpol was 1.3 ± 0.1 s−1, 12-fold lower than that opposite A, while Kd,dTTP was 8.1 ± 0.5 μM, similar to that opposite A (Figure 4B, E). For dCTP incorporation opposite Hyp, kpol was 15 ± 2 s−1, similar to that of dTTP incorporation opposite A; Kd,dCTP was 65 ± 9 μM, 9-fold higher than that of dTTP incorporation opposite A (Figure 4C, F). These data show that 6mA and Hyp reduced the incorporation efficiency (kpol/Kd,dCTP) by 14-fold and 10fold, respectively, compared with that of dTTP incorporation opposite A. However, the reasons were different: 6mA reduced the incorporation rate (kpol) but did not affect the dissociation constant (Kd,dNTP), while Hyp hardly affected the kpol but increased the Kd,dNTP. SPR Analysis of Binding of gp90 exo− to DNA Containing A, 6mA, or Hyp. To investigate whether 6mA or Hyp impacts the binding affinity of gp90 exo− to DNA, 27mer/35-mer DNA (600 RU) was immobilized on an SA chip via a biotin at the 5′ end of the primer. the DNA template contained A, 6mA, or Hyp at the 28th position. A lack of dNTP leads to a random binding of polymerase to DNA.39 The RU values at 120 s were pre-equilibrium values, and the approximate dissociation constants (Kd,DNA) were estimated as 123−157 nM for the three DNAs, indicating that 6mA or Hyp

Table 4. Approximate Dissociation Constants of gp90 exo− from Primer/Template Containing A, 6mA, or Hyp in Binary or Ternary Complex template base

dNTP

A T C 6mA T C Hyp T C

Kd, nM 137 61 99 157 71 95 123 97 61

± ± ± ± ± ± ± ± ±

14 3 7 22 7 2 13 14 6

DNA polymerase, DNA, and dNTP form a ternary complex in the presence of dNTP, in which polymerase is preferentially positioned at the 3′ end of the primer. Cdd at the 3′ end of primer blocks DNA polymerization. In the presence of dTTP (Figure 6), the approximate Kd,DNA values were estimated as 61 nM, 71 nM, and 97 nM for the ternary complex containing A, 6mA, or Hyp, respectively. The correct base pair of dTTP with A or 6mA showed lower Kd,DNA values than those between dTTP and Hyp. In the presence of dCTP (Figure 7), the approximate dissociation constants (Kd,DNA) were estimated as 99 nM, 95 nM, and 61 nM for the ternary complex containing A, 6mA, or Hyp, respectively. Similarly, the correct base pair between dCTP and Hyp gave lower Kd,DNA values than those between dCTP and A or 6mA. 844

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Figure 5. Biophysical binding of gp90 exo− to DNA containing A, 6mA, or Hyp in the absence of dNTP. (A−C) Sensorgrams for binding of gp90 exo− (10−800 nM) to 27-mer/35-mer DNA immobilized on an SA chip (600 RU) in reaction buffer B. (D−F) The approximate binding affinities of gp90 exo− to DNA were estimated by fitting the binding signal at 120 s against its corresponding gp90 exo− concentration. The solid line represents the fit curve. Standard errors were derived using Prism. Representative data from multiple experiments are shown.

Figure 6. Biophysical binding of gp90 exo− to DNA containing A, 6mA, or Hyp in the presence of dTTP. (A−C) Sensorgrams for binding of gp90 exo− (10−800 nM) to DNA immobilized on an SA chip (600 RU) in reaction buffer B containing dTTP. (D−F) The approximate binding affinities of gp90 exo− to DNA were determined by fitting the binding signal at 120 s against its corresponding gp90 exo− concentration. The solid line represents the fit curve. The standard errors were derived using Prism. Representative data from multiple experiments are shown.

binding affinity between gp90 exo− and DNA. The presence of

The Kd,DNA values of all binary complexes were higher than those of the corresponding ternary complexes (Table 4), indicating that the presence of dNTP stabilized the binding of gp90 exo− to DNA compared with the binary complexes. Furthermore, the correct base pair further enhanced the

6mA or A gave similar Kd,DNA values in a ternary or binary complex, indicating that 6mA hardly impacts the binding affinity of gp90 exo− to primer/template. 845

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Figure 7. Biophysical binding of gp90 exo− to DNA containing A, 6mA, or Hyp in the presence of dCTP. (A−C) Sensorgrams for binding of gp90 exo− (10−800 nM) to DNA immobilized on SA chip (600 RU) in reaction buffer B containing dCTP. (D−F) The approximate binding affinities of gp90 exo− to DNA were estimated by fitting the binding signal at 120 s against its corresponding gp90 exo− concentration. The solid line represents the fit curve. Standard errors were derived using Prism. Representative data from multiple experiments are shown.

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exo− to the primer/template (Table 4). Since 6mA inhibits DNA replication, 6mA may also be regarded as DNA lesion and shows the “toxicity” in DNA replication by gp90. Similarly, it was also found that, relative to unmodified A, 6mA inhibits dTTP or dUTP incorporation relative to unmodified A using either a DNA or RNA template catalyzed by the large fragment of Bst DNA polymerase.29 This inhibition is independent of template sequence. 6mA on a DNA template also inhibits dTTP or dUTP incorporation catalyzed by Klenow fragment DNA polymerase.29 Kinetic analysis shows that the efficiency of dTMP incorporation opposite 6mA is reduced. The presence of 6mA reduces the stability of DNA duplexes as determined by NMR analysis and thermodynamic measurements.42 The SPR data show that 6mA on a template has little effect on the binding affinity of gp90 exo− to DNA in binary or ternary complexes compared with unmodified A (Table 4). Furthermore, 6mA hardly impacts the binding of dTTP to the gp90 exo−-DNA complex based on our presteady-state kinetic analysis (Figure 4). Notably, 6mA reduces the burst incorporation rate (kpol). On the basis of the general dNMP incorporation mechanism,43 the burst incorporation step consists of conformational change and phosphodiester bond formation. The amino acid sequence of gp90 is highly homologous to those of other A-family T7 DNA polymerase (Pol T7−) and DNA Pol I31. These A-family polymerases always show that the conformational change step is relatively slow and limits the overall correct dNMP incorporation.44−46 Therefore, 6mA may inhibit one of the steps involved in the burst incorporation rate constant, i.e., a conformational change or chemistry step. Furthermore, molecular dynamics shows that 6mA on a template tends to enter into and is restrained in the minor groove recognition region of Bst DNA polymerase, thus decreasing the conformational flexibility of DNA

DISCUSSION 6mA as an epigenetic marker plays important roles in regulation of various biological processes.23,40,41 However, the effect of 6mA on DNA replication is little identified. It has been newly found that 6mA is also present in Pseudomonas aeruginosa Phage PaP1 genome.30 How 6mA affects DNA replication by PaP1 DNA polymerase should be explored. Previously, we have identified that gp90 is the only DNA polymerase found in PaP1, and it can be considered as a model of A-family DNA polymerases.31 Gp90 exo− could bypass 8oxoG error-free with reduced incorporation efficiency.32 O6MeG, an alkylation lesion, partially inhibits prime extension by gp90 exo−, leading to a 67-fold priority in dTTP misincorporation rather than correct dCTP incorporation.33 Gp90 exo− preferentially incorporates dATP opposite an abasic site via the A-rule, independent of the 5′-next template sequence.1 In this work, we found that DNA polymerization by gp90 exo− is partially inhibited by 6mA or Hyp on the template (Figure 2). Steady-state kinetic analysis shows that 6mA reduces dTTP incorporation efficiency (kcat/Km) by 10-fold and increases the misincorporation frequencies by 10-fold (Table 2). dCTP is preferentially incorporated opposite Hyp, resulting in the mutation from an A:T pair to a G:C pair. 6mA also reduces the next-base extension efficiency by 100-fold and loses the priority in extension beyond the 6mA:T pair rather than the 6mA:C mispair (Table 3). Gp90 exo− also preferentially extends beyond Hyp:C rather than the Hyp:T pair. 6mA and Hyp reduce the burst incorporation efficiency (kpol/Kd,dNTP) by 14-fold and 10-fold, respectively, compared with that of the dTTP incorporation opposite A, because 6mA reduces the incorporation rate (kpol) and Hyp increases the dissociation constant (Kd,dNTP; Figure 4). The presence of 6mA or A gives similar Kd,DNA values in a ternary or binary complex, indicating that 6mA hardly impacts the binding affinity of gp90 846

DOI: 10.1021/acs.chemrestox.8b00348 Chem. Res. Toxicol. 2019, 32, 840−849

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Chemical Research in Toxicology polymerase.29 Taken together, 6mA hardly influences the binding of dTTP to the polymerase−DNA complex but highly possibly affects the conformational change or chemistry step and reduces dTTP incorporation efficiency. The methyl group at 6mA does not affect H-bond formation with the incoming dTTP, thus showing moderate effects on primer extension and dTTP incorporation. Differently, the methyl group at O6-MeG, 3-methylcytosine (3mC), or 1methyladenine (1mA) disrupts H-bond formation and shows significantly harmful effects on DNA replication. O6-MeG obviously inhibits primer extension by gp90 exo−, reduces dCTP incorporation efficiency by a 106-fold magnitude compared with unmodified G, and results in a 67-fold preferential incorporation of dTTP rather than dCTP.33 3mC and 1mA are highly toxic and mutagenic, which disrupts the hydrogen bonding between base pairs.47 In E. coli, these lesions block DNA replication, potentially because of the inhibition of B-family replicative Pol III.48 3mC also inhibits primer extension by Sulfolobus solfataricus Y-family Dpo4 and human B-family Pol δ and blocks extension by X-family Pols β and λ.49 1-MeA lesion impairs Watson−Crick base pairing and blocks normal DNA replication. Translesion DNA synthesis across 1-MeA in human cells occurs in a highly error-prone fashion via hPol η and ι.50 Previously, we have studied nucleotide incorporation opposite Hyp via Pol T7− and Dpo4.26 All the kinetic analyses show that Hyp is similar to G and dCMP, similar to the results obtained from gp90 exo−. Additionally, the dissociation constants of dCTP from the polymerase−DNA complex were higher for Hyp than for G for all gp90 exo−, Pol T7−, and Dpo4,1,26,32,33 mainly because G has three H-bonds with dCTP but Hyp has only two. Thus, all the results show that Hyp is an analogue of G but has a weaker H-bond formation ability with dCTP. Abasic sites are produced at a rate of ∼50 000/cell/day.51 On the basis of 3 billion base pairs in human cells, the percentage of abasic sites is approximately 0.0017%. 6mA is present in the human genome, accounting for ∼0.051% of the total adenines.20 The abundance of 6mA in Drosophila DNA is in the range of 0.001−0.07% (6mA/dA) during embryonic development.15 The PaP1 genome (91 715 bp) contains 51 6mA,30 accounting for ∼0.2% (6mA/dA). Since gp90 is the sole DNA polymerase found in PaP1,31 6mA at a pretty high abundance in the PaP1 genome inevitably inhibits DNA replication by gp90 and the overall DNA replication efficiency of PaP1. PaP1 is a lytic phage of P. aeruginosa (Pa). The lysis of Pa by PaP1 depends on the relative efficiency of Pa proliferation and PaP1 propagation. 6mA in PaP1 could reduce PaP1 propagation and inhibit the lysis of its host Pa. Additionally, the distribution of 6mA in the PaP1 genome is not random but is concentrated on the sequence of 5′GGACT-3′, where A could be methylated to form 6mA.30 Thus, the formation of 6mA should not be stochastic but be catalyzed by some unknown endogenous methylation enzymes that rely on this specific DNA sequence. Therefore, the epigenetic functions of 6mA on biological processes, such as PaP1 propagation, infection of its host Pa, and interaction between PaP1 and its host, remain to be further discovered. In conclusion, we revealed the nucleotide incorporation opposite 6mA or Hyp by gp90 exo−. Primer extension by gp90 exo− is partially inhibited by 6mA or Hyp on the template. 6mA reduces dTTP incorporation efficiency and next-base extension efficiency. dCTP is preferentially incorporated

opposite Hyp. 6mA reduces the burst incorporation rate (kpol), while Hyp increases the dCTP dissociation constant (Kd,dNTP). Biophysical binding assays show that 6mA or Hyp does not affect the binding of DNA polymerase to DNA in binary or ternary complexes. This work provides new insight in the inhibited effects of the epigenetically modified 6mA on DNA replication by DNA polymerase in PaP1.

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AUTHOR INFORMATION

Corresponding Authors

*Lead contact. (H.Z.) E-mail: [email protected]. *(Z.S.) E-mail: [email protected]. ORCID

Huidong Zhang: 0000-0001-9810-549X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the China Key Research and Development Program [2017YFC1002002], the Fundamental Research Funds for the Central Universities, National Natural Science Foundation of China [31370793, 81422041], and the Youth 1000 Talent Plan. Acknowledgment is given for the facility support by the Central Laboratory of West China College of Public Health at Sichuan University.

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ABBREVIATIONS Gp90 exo−, exonuclease-deficient gene 90 protein; 6mA, N6methyladenine; Hyp, hypoxanthine; Pa, P. aeruginosa; PaP1, Pseudomonas aeruginosa phage; Pol, polymerase; dNTP, deoxyribonucleoside triphosphate; SA, streptavidin; RU, response unit(s); SPR, surface plasmon resonance

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REFERENCES

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