Epigenetic DNA Modification N6-Methyladenine Inhibits DNA

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Epigenetic DNA Modification N-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 Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00348 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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Chemical Research in Toxicology

Epigenetic DNA Modification N6-methyladenine Inhibits DNA Replication by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Bianbian Li1,2, Ke Du2, Shiling Gu2, Jiayu Xie2, Tingting Liang2, Zhongyan Xu2, Hui Gao1, Yihui Ling3, Shuguang Lu4, Zhen Sun1,* and Huidong Zhang2,5,*

1 School

2 Key

of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China

Laboratory of Environment and Female Reproductive Health, West China School of

Public Health & West China Fourth Hospital, Sichuan University, Chengdu, China 3

Institute for Chemical Carcinogenesis, Guangzhou Medical University, Xinzao, Panyu

District, Guangzhou, China 4

Department of Microbiology, College of Basic Medical Science, Third Military Medical

University, Chongqing, China 5

Lead contact

* To whom correspondence should be addressed: Huidong Zhang, Key Laboratory of Environment and Female Reproductive Health, West China School of Public Health & West China Fourth Hospital, Sichuan University, No.17 People's South Road, Chengdu, 610041,

China. E-mail: [email protected]. Tel: +086 288550 1580; Fax: +86 28 8850 1580. Zhen Sun, School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China, E-mail: [email protected].

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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−. 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 6mA:T pair rather than 6mA:C mispair and preferred to extend beyond Hyp:C rather than 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 complex. This work provides new insight in the inhibited effects of epigenetic modification 6mA on DNA replication in PaP1.

Keywords: Pseudomonas aeruginosa Phage PaP1; DNA polymerase; DNA replication; N6-methyladenine (6mA); hypoxanthine (Hyp); Steady-state and Pre-steady-state kinetics.



INTRODUCTION

Efficient and accurate DNA replication is very important to keep genomic integrity during cell division and proliferation. However, various exogenous and endogenous factors produce diverse DNA lesions or modifications, which damage DNA replication machinery1, 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 cancer3.

DNA lesions are produced through various chemical reactions, such as oxidation, alkylation (which may involve cross-linking), deamination, photo-addition, coordination, and hydrolysis3. Genomic DNA can also be methylated by various endogenous methyltransferase 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 pairing6-8. Differently, N1-methyladenine (1mA) and N3-methylcytosine (3mC) are considered as DNA lesions because these modifications disrupt H-bond formation9.

6mA is primarily present in prokaryotes10. Recently, 6mA was also found in eukaryote genomes11, including green algae12, nematodes13, fungi14, insects15, vertebrates16, mouse embryonic stem cells17, Arabidopsis thaliana18, rice19, zebrafish and pig4, and human20. The genomic distribution of 6mA varies among different species. 6mA levels are relatively high in unicellular eukaryotes but very low in metazoans21. About 70% of 6mA sites are located in genes and concentrated around the transcription start sites22.


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6mA is generated by methylation of Adenine (A) catalyzed by MT-A70 family methylases in eukaryotes23, 24 (Fig. 1). 6mA can be oxidized by AlkB family enzymes to form 6-hydroxymethyladenine (6hmA), which releases its formaldehyde group to produce A24, 25. Alternatively, 6mA can also be deaminated to generate hypoxanthine (Hyp, Fig. 1), which is subsequently removed by base excision repair via AlkA family enzymes, and then the correct dATP is incorporated before DNA ligation24, 26. Adenine can also be converted to Hyp via oxidative deamination during inflammation and oxidation by nitric oxide27, 28. In C. elegans, DNA methyltransferase (DAMT-1) and demethylase (NMAD-1) regulate 6mA levels and crosstalk between methylations of adenines13.

In prokaryotes, 6mA is signaling or epigenetic mark and distinguishes self from foreign DNA6. In bacteria, 6mA plays important roles in regulation of DNA mismatch repair, chromosome replication, cell cycle regulation, transcription, and restriction-modification12. In eukaryotes, 6mA was also found as heritable epigenetic modification13. During early embryo development in metazoans, the elevated levels of 6mA serves as an important epigenetic mark to control transcription21. In green alga Chlamydomonas, 6mA accumulation is related to gene activation12. In vitro, 6mA hinders DNA synthesis on DNA or RNA template by a large fragment of Bst DNA polymerase29.

Recently, 6mA was also found in the genome of Pseudomonas aeruginosa phage (PaP1)30. PaP1 genome contains 91,715 base pairs and 51 6mA. However, whether and how 6mA affects DNA replication in PaP1 are unknown. Recently, we have identified that DNA polymerase of PaP1, gp90, an A-family processive DNA polymerase containing 3′→5′



exonuclease activities on ssDNA and dsDNA31. Exonuclease-deficient gp90 exo− can error-free bypass 8-oxoG32, was partially inhibited by an alkylation lesion, O6-MeG33, and was completely blocked by an abasic site1. In this 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, 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.

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 previously1. 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 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 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


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One™ software33, 34.

Steady-state kinetic analysis of single-nucleotide incorporation and next-base extension. Reactions were performed using 32P-labeled 24-mer or 25-mer primer/35-mer template containing A, 6mA, or Hyp (Table 1) with the molar ratio of gp90 exo− to DNA substrate < 10% in buffer A at 37°C26. The extent of primer extension was controlled < 0.20% by adjusting polymerase concentrations and reaction time26. 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 incorporation efficiency (kcat/Km) of each misincorporated dNTP by that of dCTP35, 36. The standard errors were derived using Prism. Pre-steady-state kinetic analysis of nucleotide incorporation. Pre-steady-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 dNTP incorporation in the burst phase and in the steady-state phase. All parameters and standard errors were derived using Prism.

𝑦 = 𝐴(1 ― e𝑘pt) + 𝑘ss𝑡

(1)

Where y is product concentration, nM; A is burst amplitude, nM; kp is burst rate, s−1; t is time, s; kss is 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)

(2)

𝑘obs = 𝑘pol[dNTP]/([dNTP] + 𝐾d,dNTP)

(3)

Where kobs is burst incorporation rate, s−1; kpol is the maximal burst rate, s−1; 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 primer/template containing A, 6mA, or Hyp was determined using a Biacore T200 (Uppsala, Sweden) as described previously37, 38. A 27-mer 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 (Table1). DNA was immobilized on SA chip (600 RU). Gp90 exo− (10-800 nM) were 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 sec. Binding signals at 120 sec versus gp90 exo− concentrations were fitted to Eq. 4.

𝑌 = 𝐵 × 𝑅𝑈/(𝐵 + 𝐾d)

(4)

Where Y is binding response signal at 120 sec, RU; RU is the binding signal at 120 sec, 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.

RESULTS


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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 24-mer primer to 35-mer product on unmodified template; while this extension was relatively inhibited by 6mA and Hyp (Fig. 2). The percentages on the top of each gel show the conversion of primer to 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−.

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 10-5-10-6. The kcat values of the three incorrect dNTPs were decreased 6-16 folds relative to that of dTTP; while their Km values were thousand-fold higher than that of dTTP. Opposite 6mA, dTTP was preferentially incorporated. The misincorporation frequencies of three incorrect dNTPs were 10-4-10-5. The efficiency of dTTP incorporation opposite 6mA was 11-fold lower than that opposite A. Differently, dCTP was preferentially incorporated opposite Hyp, four orders of magnitude more efficiently than other three dNTPs. The 5′ next nucleotide was T, precluding the possibility that dCTP was directly incorporated opposite the next nucleotide T through -1 frameshift deletion. Taken together, 6mA on template reduced dTTP incorporation efficiency and Hyp on template preferred to 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 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 A:T pair (primer:template) than A:C mispair, due to similar kcat values but significantly decreased Km value. For template 6mA, dATP incorporation opposite next base T was 100-fold less efficient beyond 6mA:T than beyond A:T pair, indicating that 6mA inhibited the next-base extension. Additionally, the extension efficiency was only 15-fold preferential in extension beyond 6mA:T pair rather than 6mA:C mispair, showing that 6mA greatly reduced the priority in extension beyond correct pair rather than mispair. The extension efficiency was about 1300-fold higher beyond Hyp:C rather than Hyp:T pair. Therefore, gp90 exo− preferentially extended beyond 6mA:T pair or Hyp:C pair, but inhibited the extension efficiency and lost priority in beyond 6mA:T pair, and was completely prefer to extension beyond Hyp:C rather than Hyp:T pair.

Pre-steady-state kinetic analysis. If DNA is molar excessive relative to DNA polymerase, correct dNTP incorporation generally shows burst phase and steady-state phase for most DNA polymerases33. 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 phase32, 33. To determine the burst kinetic parameters of dNTP incorporation opposite A, 6mA or Hyp by gp90 exo−, a molar excess of DNA than gp90 exo− was used. dTTP among four dNTPs was preferentially incorporated opposite A or 6mA, showed a biphasic feature with the burst rate kp of 14 s−1 or 0.81 s−1, respectively (Fig. 3A, B). The incorporation of three incorrect dNTPs exhibited linear phases. Incorporation of dCTP


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opposite Hyp was preferential relative to other three dNTPs and showed a biphasic phase with a kp value of 10 s−1 (Fig. 3C). Incorporations of 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 -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 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 (Fig. 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 kpol value of 16 ± 1 s−1 and Kd,dTTP of 7.3 ± 0.5 μM (Fig. 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 (Fig. 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 (Fig. 4C, F). These data show that 6mA and Hyp reduced the incorporation efficiency (kpol/Kd,dCTP) by 14-fold and 10-fold, 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 affect 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 impact the binding affinity of gp90 exo− to DNA, 27-mer/35-mer DNA (600 RU) was immobilized to a SA chip via a biotin at the 5’ end of primer. DNA template contained A, 6mA, or Hyp at the 28th-position. Lack of dNTP leads to a random binding of polymerase to DNA39. 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 hardly impact the binding of gp90 exo− to DNA (Table 4, Fig. 5).

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 primer. Cdd at the 3' end of primer blocks DNA polymerization. In the presence of dTTP (Fig. 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 that between dTTP and Hyp. In the presence of dCTP (Fig. 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 that between dCTP and A or 6mA.

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


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further enhanced the binding affinity between gp90 exo− and DNA. The presence of 6mA or A gave similar Kd,DNA values in ternary or binary complex, indicating that 6mA hardly impact the binding affinity of gp90 exo− to primer/template.

DISCUSSION

6mA as an epigenetic marker plays important roles in regulation of various biological processes23, 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 genome30. 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 polymerases31. Gp90 exo− could error-free bypass 8-oxoG with reduced incorporation efficiency32. O6-MeG, an alkylation lesion, partially inhibits prime extension by gp90 exo−, leading to a 67-fold priority in dTTP misincorporation rather than correct dCTP incorporation33. Gp90 exo− preferentially incorporates dATP opposite an abasic site via the A-rule, independent of the 5'-next template sequence1.

In this work, we found that DNA polymerization by gp90 exo− is partially inhibited by 6mA or Hyp on template (Fig. 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 A:T pair to G:C pair. 6mA also reduces the next-base extension efficiency by 100-fold and loses the priority in extension beyond 6mA:T pair rather than 6mA:C mispair



(Table 3). Gp90 exo− also preferentially extends beyond Hyp:C rather than 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 dTTP incorporation opposite A, because 6mA reduces the incorporation rate (kpol) and Hyp increases the dissociation constant (Kd,dNTP) (Fig. 4). The presence of 6mA or A gives similar Kd,DNA values in ternary or binary complex, indicating that 6mA hardly impact the binding affinity of gp90 exo− to 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 DNA or RNA template catalyzed by the large fragment of Bst DNA polymerase29. This inhibition is independent on template sequence. 6mA on DNA template also inhibits dTTP or dUTP incorporation catalyzed by Klenow fragment DNA polymerase29.

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 measurements42. The SPR data shows that 6mA on 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 impact the binding of dTTP to gp90 exo−-DNA complex based on our pre-steady-state kinetic analysis (Fig. 4). Notably, 6mA reduces the burst incorporation rate (kpol). Based on the general dNMP incorporation mechanism43, the burst incorporation step is consisted of conformational change


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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 incorporation44, 45, 46. Therefore, 6mA may inhibits one of the steps involved in the burst incorporation rate constant, i.e., conformational change or chemistry step. Furthermore, Molecular dynamics shows that 6mA on 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 polymerase29. Taken together, 6mA hardly influence the binding of dTTP to polymerase-DNA complex, but high 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, dTTP incorporation. Differently, the methyl group at O6-MeG, 3-methylcytosine (3mC), or 1-methyladenine (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 106-fold magnitude compared with unmodified G and results in 67-fold preferential incorporation of dTTP rather than dCTP33. 3mC and 1mA are highly toxic and mutagenic, which disrupt the hydrogen bonding between base pairs47. In E. coli, these lesions block DNA replication, potentially because of the inhibition of B-family replicative Pol III48. 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 λ49. 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 by hPol η and 50. Previously, we have studied nucleotide incorporation opposite Hyp by Pol T7- and Dpo426. All the kinetic analysis shows that Hyp is similar to G and dCMP, similar to the results obtained from gp90 exo−. Additionally, the dissociation constants of dCTP from polymerase-DNA complex were higher for Hyp than for G for all gp90 exo−, Pol T7- and Dpo41, 26, 32, 33, highly 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/day51. Based on 3 billion base pairs in human cells, the percentage of abasic site is approximate 0.0017%. 6mA is present in human genome, accounting for ~ 0.051% of the total adenines20. Abundance of 6mA in Drosophila DNA is in the range of 0.001% - 0.07% (6mA/dA) during embryonic development15. PaP1 genome (91,715 bp) contains 51 6mA30, accounting for ~ 0.2% (6mA/dA). Since gp90 is the sole DNA polymerase found in PaP131, 6mA at a pretty high abundance in PaP1 genome inevitably inhibits DNA replication by gp90 and the overall DNA replication efficiency of PaP1. PaP1 is 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 PaP1 genome is not random, but is concentrated on the sequence of 5′-GGACT-3′, where A could be methylated to form 6mA30. 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


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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 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 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.

FUNDING INFORMATION AND ACKNOWLEDGEMENTS This work was supported by 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 for the facility supports by Central Laboratory of West China College of Public Health at Sichuan University.

NOTES

No conflicts of interest with the contents of this article.

ABBREVIATIONS LIST



Gp90 exo−, exonuclease-deficient gene 90 protein; 6mA, N6-methyladenine; 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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Page 22 of 35

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TABLES

Table 1. Oligodeoxynucleotieds. 24-mer

5′-TCGCATAGATCTCAGGTCAAGTAC-3′

25T-mer

5′-TCGCATAGATCTCAGGTCAAGTACT-3′

25C-mer

5′-TCGCATAGATCTCAGGTCAAGTACC-3′

27-mer

5′-biotin-TTTTCGCATAGATCTCAGGTCAAGTACdd-3′

35-mer

3′-AGCGTATCTAGAGTCCAGTTCATGA*TCGCTTACGA-5′

A*: A, 6mA, or Hyp



Page 24 of 35

Table 2. Steady-state kinetic parameters of single-nucleotide incorporation opposite A, 6mA, or Hyp by gp90 exo−a. Misincorpo Primer kcat, kcat/Km, Efficiency dNTP K , μM -ration m, dNTP template ×10-3 s-1 relative to A:dTTP μM-1s-1 frequencyb

5′-C 3′-GATC

5′-C 3′-G6mATC

5′-C 3′-GHypTC

dTTP

27 ± 0.01c

(1.2 ± 0.1) ×10-3

23

-

dATP

4.2 ± 0.04

6.8 ± 1.3

6.2 × 10-4

2.7 × 10-5

3.7 × 104-fold less

dGTP

1.7 ± 0.01

10 ± 1.5

1.7 × 10-4

7.4 × 10-6


dCTP

2.5 ± 0.01

2.8 ± 0.3

8.9 × 10-4

3.9 × 10-5


dTTP

5.9 ± 0.01

(2.8 ± 0.4) × 10-3

2.1

-

dATP

3.7 ± 0.01

2.4 ± 0.1

1.5 × 10-3

7.1 × 10-4


dGTP

2.0 ± 0.02

14 ± 2.3

1.4 × 10-4

6.7 × 10-5


dCTP

1.1 ± 0.01

9.9 ± 1.4

1.2 × 10-4

5.7 × 10-5


dTTP

3.4 ± 0.01

0.44 ± 0.02

7.8 × 10-3

5.2 × 10-4


dATP

3.5 ± 0.01

0.56 ± 0.07

6.3 × 10-3

4.2 × 10-4


dGTP

0.8 ± 0.01

0.47 ± 0.07

1.7 × 10-3

1.1 × 10-4


dCTP

3.6 ± 0.01

(2.4 ± 0.2) × 10-4

15

-

1

11-fold less

1.5-fold less

The extent of incorporation was controlled < 20% by adjusting polymerase concentration and reaction time. b Misincorporation frequency is defined as (k /K ) cat m incorrect dNTP/(kcat/Km) correct dTTP or dCTP. c The standard errors were derived using Prism. a


Page 25 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Steady-state kinetic parameters for next-base extension beyond A, 6mA, or Hyp by gp90 exo−. Primer template 5′-CX 3′-GATC 5′-CX 3′-G6mATC 5′-CX 3′-GHypTC

Primer X

kcat, × 10-3 s-1

Km, dATP, μM

kcat/Km, × 10-3 μM-1 s−1

T

2.2 ± 0.08

0.20 ± 0.05

12

C

0.80 ± 0.02

87 ± 11

9.2 × 10

1300-fold less

T

4.1 ± 0.04

34 ± 7

0.12

100-fold less

C

1.7 ± 0.01

220 ± 35

8.0 × 10

1500-fold less

T

1.1 ± 0.05

110 ± 15

0.01

1200-fold less

C

1.3 ± 0.05

0.10 ± 0.03

13

0.90-fold less

Efficiency relative to A:T pair 1

-3

-3

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



Page 26 of 35

Table 4. Approximate dissociation constants of gp90 exo− from primer/template containing A, 6mA, or Hyp in binary or ternary complex. Template base A

6mA

Hyp

dNTP

Kd, nM

T C T C T C

137 ± 14 61 ± 3 99 ± 7 157 ± 22 71 ± 7 95 ± 2 123 ± 13 97 ± 14 61 ± 6


Page 27 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURE LEGENDS

Figure 1. Structure illustration of adenine (A), N6-methyladenine (6mA), and hypoxanthine (Hyp), and scheme of their conversions. 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 substrate and 35-mer product. Percentages on top depict the conversions of primer to 35-mer product. Representative data from multiple experiments are shown. Figure 3. Pre-steady-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 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.

Figure 4. Pre-steady-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. 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 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 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. 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.


Page 28 of 35

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FIGURES

A

6mA NH2

N

N DNA

N

N

Hyp

CH3 HN

N

O

N

Methylation N

N

NH

Deamination

N

DNA

Deamination Deglycosylation/depurination

Figure 1


N DNA

N


3% 6% 11% 26%

2% 4% 6% 16%

Page 30 of 35

2% 5% 9% 17%

35-mer

24-mer

Gp90 exo- : 0 2

5 10 20

A

0

2 5 10 20

6mA

Figure 2


0

2 5 10 20 nM

Hyp

Page 31 of 35

A = 12 ± 0.8 KP = 0.80 ± 0.010 s-1

A = 20 ± 1.0

A

B

A 40

dTTP

C

6mA 25

30

Product, nM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dTTP

50

20

kp = 14 ± 3

s-1 dCTP dATP dGTP

0 0

10

20

30

kp = 0.81 ± 0.01

10

10

40

50

公公公公公公公公公公

dCTP

Best-f it v alues

40

15

20

I Hyp

30

s-1

5 0 0

10

20

30

40

kp = 10 ± 1.8

s-1 dATP dTTP dGTP

10 0

50

Time, s

Figure 3


36.72

k

10.13

kss

0.2880

Std. Error

20

dATP dCTP dGTP

A

0

10

20

30

40

50

A

1.676

k

1.935

kss

0.08632


A dTTP A ++ dTTP

A

6mA++dTTP dTTP 6mA

B

50

20

Product, nM

I++ dCTP Hyp dCTP

C

30

25

40

20

15 10

30 20

10

5

10

0 0.0

0.5

Kpol = 16 ± 1.0 s-1 K1.0 7.3 ± 2.0 0.50 M 0 0 d, dTTP = 1.5

D

1

E

kobs, s-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

2

3

Kpol = 1.3 ± 0.050 s-1 Kd, dTTP = 8.1 ±0 0.50 M

4

0.0

5

1.5

15

10

1.0

10

kpol = 16 ± 1 s-1 Kd, dTTP = 7.3 ± 0.5 μM

0 0

10

20

30

dTTP, M

kpol = 1.3 ± 0.1 s-1

0.5

40

50

0

20

40

60

1.5

2.0

kpol = 15 ± 2 s-1 Kd, dCTP = 65 ± 9 μM

5

Kd, dTTP = 8.1 ± 0.5 μM

0.0

1.0

F

Time, s

15

5

0.5

Kpol = 15 ± 2.0 s-1 Kd, dCTP = 65 ± 9.0 M

80

100

dTTP, M

Figure 4


0 0

50

100

dCTP, M

150

200

Page 33 of 35

A

B

Gp90exo− AA++gp90

Response, RU

Stop

Stop

8000

C

6mA + Gp90 6mA + gp90 exo− 8000

4000

Inject

4000

Inject Kd, DNA 2000 = 140 ± 16 nM

2000

2000

50

100

150

200

D

0

E 8000 6000 4000 2000

Kd, DNA = 137 ± 14 nM

0

50

100

150

200

Time, s

100

200

300

400

500

600

0

F

8000

Kd, DNA= 120 ± 14 nM

6000

4000

4000

2000

Kd, DNA = 157 ± 22 nM

50

100

150

200

8000

6000

Kd, DNA = 123 ± 13 nM

2000 0

0 0

Inject

0

0

0

Stop

6000

6000 4000

Gp90exo− Hyp I++gp90 8000

6000

0

Response, RU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

200

400

600

800

Gp90,exo nM-, nM gp90

Figure 5


0

200

400

600

800


A

+ Gp90 A +Agp90 exo+- dTTP + dTTP 6000

B

6mA + Gp90 + -dTTP 6mA + gp90 exo + dTTP

Response(RU)

C

- + dTTP Gp90exo + dTTP HypI++gp90

6000

8000

Stop

Stop

Stop 6000

4000

4000

4000 2000

Inject

2000

0 0

D

50

100

Kd, DNA200 = 60 150

2000

Inject

E

50

100

Kd,150 ± 7.0 nM 0 DNA= 68 200

Kd,DNA= 96 ± 14 nM 50

100

150

200

F

Time, s

6000

6000

4000

Inject

0

0

± 3.0 nM 0 8000

6000

Response(RU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 35

4000

4000 2000

2000

Kd, DNA = 71 ± 7 nM

2000

Kd, DNA = 61 ± 3 nM 0

Kd, DNA = 97 ± 14 nM 0

0 0

100

200

300

400

500

600

0

200

400

600

800

Gp90, gp90 exo-nM , nM

Figure 6


0

200

400

600

800

Page 35 of 35

A

A + gp90 exo- + dCTP

Response, RU

6000

2000

6mA + Gp90 + -dCTP 6mA + gp90 exo + dCTP

8000

4000

Inject

Stop

150

4000

200

Inject

0

E

6000

4000

2000

Kd, DNA = 99 ± 7 nM

2000

100

150

200

400

600

800

Kd, DNA= 59 ± 5.0 nM 0

200

8000

F 8000

6000

6000

4000

4000 2000

Kd, DNA = 95 ± 2 nM

50

100

150

200

Kd, DNA = 61 ± 6 nM

0

0 0

Inject

Kd, DNA= 96 ± 3.0 nM0 50

Time, s

2000

0

Stop

4000

0

100

- + dCTP Gp90exo + dCTP Hyp I++gp90

8000 6000

2000

50

C

6000

Kd, DNA=97 ± 8.0 nM 0

D

B

Stop

0

Response, RU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

200

400

600

800

GP90, nM gp90 exo-, nM

Figure 7


0

200

400

600

800

Epigenetic DNA Modification N6-Methyladenine Inhibits DNA

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