Type II Restriction of 2-Aminoadenosine (dZ)-Modified DNA and Production of dZ-Modified Plasmid in E. coli

Abstract

The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host-encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Here, we report the activity results of 147 REases on dZ-modified PCR DNA. Among the enzymes tested, 53% are resistant or partially resistant, and 47% are sensitive when their restriction sites contain one to six modified bases. Sites with four to six dZ substitutions are most likely to resist Type II restriction. Our results support the notion that dZ-modified phage genomes evolved to combat host-encoded restriction systems. dZ-modified DNA can also reduce phage T5 exonuclease degradation, but has no effect on RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into an E. coli plasmid, the level of dZ incorporation reached 19–20% of adenosine positions. dZ levels further increased to 29–44% with co-expression of a DNA polymerase gene from the same phage. High levels of dZ incorporation in recombinant plasmid are possible by co-expression of purZ, mazZ, datZ and phage DNA helicase, dpoZ (DNA polymerase) and ssb (single-stranded DNA binding protein SSB). This work expands our understanding of the dZ modification of DNA and opens new avenues for engineering restriction systems and therapeutic applications.

1. Introduction

In the biological arms race between bacterial hosts and phages, phages evolved DNA base modifications to counteract host-encoded restriction systems [1,2,3,4] (reviewed in [5,6,7]). DNA base modifications can occur on all four canonical bases. Examples include cytosine modifications such as 5-methylcytosine (5mC) in phage Xp12, 5-hydroxymethylcytosine (5hmC) in phage T4gt, and glucosylated 5hmC in wild-type T4; guanine modification with deoxyarchaeosine (dG⁺) in phage 9g; thymidine modification with α-putrescinylthymidine (putT) in phage φW-14; and adenine modifications such as N6-carbamoylmethyl (glycinamide) in phage Mu (6-NcmdA) and 2-aminoadenosine (dZ) in cyanophage S-2L, which can form three hydrogen bonds with thymidine [6] (reviewed in [7]). It has been previously demonstrated that phage genomes containing dZ bases are more resistant to Type II restriction compared to unmodified DNA nucleotides [8,9,10]. Although a limited number of REases have been tested on dZ-modified phage DNA, these results may be confounded by additional modification systems encoded by the host during phage DNA replication. It is known that bacterial hosts can harbor multiple R-M systems or orphan methylases (MTases) [11]. In some cyanobacteria, R-M systems and orphan M systems can reach 40–50 [3]. To assess the effect of dZ in Type II restriction, we synthesized dZTP using an adapted protocol and used it in PCR to generate DNA substrates containing dZ as the only modification. These substrates were then subjected to restriction digestion assays. In addition, we examined exonuclease digestions of the dZ-modified DNA to monitor for reduced degradation rates. Indeed, a few exonucleases were less efficient at digesting dZ-modified substrates, which may have implications for DNA stability in practical applications such as gene therapy, gene targeting, and DNA vaccines.

It has been shown that through expression of three phage genes in E. coli (purZ, datZ, mazZ), dZ-containing plasmid DNA can be generated with up to 20% dZ replacing adenosine [12]. We aim to use dZ-containing plasmids to screen for dZ-dependent REases, which necessitates further enhancement in dZ incorporation levels in these plasmids. Moreover, dZ-containing plasmids may be useful in certain therapeutic applications, requiring low cost of production of such modified DNA in E. coli. To increase dZ levels in E. coli plasmids, we investigated the expression of three genes from Salmonella phage PMBT28 (purZ (2-adenylosuccinate synthetase), datZ (dATP triphosphohydrolase, dATPase), and mazZ ((d)GTP-specific diphosphohydrolase) cloned into a T7 expression vector pET21b. Because the codon usage of Salmonella is more compatible with E. coli expression systems, dZ levels may be increased through expression under IPTG induction. In addition, the DNA polymerase gene from Salmonella phage PMBT28 was cloned into the same plasmid as dZ modification genes or co-expressed in a compatible plasmid to further increase dZ incorporation. We also assessed co-expression of six phage genes in E. coli to enhance dZ incorporation.

2. Materials and Methods

2.1. dZTP Chemical Synthesis

Unless otherwise noted, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. 2-amino-2′deoxyadenosine was purchased from Toronto Research Chemicals (North York, ON, Canada). Analytical reverse-phase HPLC was performed on an Agilent 1200 series LC/MS system equipped with a G1315D diode array UV-Vis detector and a 6120 quadrupole LC/MS detector in both positive (+ESI) and negative mode (−ESI). LC was performed on a Phenomenex (Torrance, CA, USA) Clarity Oligo-XT C18 column (4.6 × 100 mm, 2.6 µm, 100 Å) with a gradient mobile phase consisting of 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol and 4 mM hexylamine in water and methanol. Relative abundance of product to reactants was determined by UV. Preparative-scale HPLC was done on an Agilent 1290 Infinity II, with a Phenomenex Gemini NX C-18 column (30 × 100 mm, 10 µm, 110 Å) using a gradient mobile phase of 200 mM triethylammonium bicarbonate in water and acetonitrile. Preparative-scale anion exchange was done on an AKTA Pure 150 FPLC from Cytiva (Marlborough, MA, USA), with a 50 × 250 mm column containing Ceramic HyperDF DEAE resin from Pall (Port Washington, NY, USA), with a gradient mobile phase of water and 1.0 M triethylammonium bicarbonate in water, pH 7.4. Nuclear Magnetic Resonance (NMR) and High-Resolution MS (HRMS) were performed by Novatia LLC (Newtown, PA, USA).

In a round-bottom flask, 1.1 g (4.1 mmol, 1.0 eq.) of 2-amino-2′-deoxyadenosine was dried in vacuo overnight over phosphorus pentoxide. In a separate flask, 30 mL of trimethyl phosphate was dried over molecular sieves for two days. Dried 2-amino-2′deoxyadenosine was dissolved in 30 mL of trimethyl phosphate, then solution was cooled on ice to 0 °C, under inert argon atmosphere. Then, 0.76 mL (8.2 mmol, 2.0 eq.) of phosphorus oxychloride was added to the solution over a period of 1 min with vigorous stirring. The reaction was stirred at 0 °C until high conversion was confirmed by LC-MS analysis. An ice-cold solution of tributylammonium pyrophosphate (7.52 g, 16.5 mmol, 4.0 eq.) and tributylamine (3.9 mL, 16.5 mmol, 4.0 eq.) in dry DMF was added with vigorous stirring at 0 °C. The reaction was monitored by LC-MS and quenched with 500 mL 0.2 M triethylammonium bicarbonate. The crude reaction product was diluted to 1 L with dH₂O and then purified by DEAE. The isolated product fractions were combined, evaporated to dryness, co-evaporated with methanol twice, and purified by reverse-phase HPLC. Product fractions were combined, evaporated to dryness, then co-evaporated three times with 20 mL methanol to yield a clear oil corresponding to 1.2 mmol of dZTP as a triethylammonium salt (28% yield). The purity of dZTP was determined by LC-MS to be 98%. ¹H NMR (500 MHz, D₂O): δ 8.09 (s, 1H), 6.23 (m, 1H), 4.71 (m, 1H) 4.20 (m, 1H), 4.13 (m, 2H), 2.68 (m, 1H), 2.45 (ddd, J_gem = 13.9, J_2′b1′ = 6.3, J_2′b,3 = 3.5, 1 H). ¹³C NMR (126 MHz, D₂O): δ 158.6, 154.7, 137.8, 137.7, 112.4, 85.6, 83.2, 71.1, 65.5, 38.8. ³¹P NMR (202 MHz, D₂O): δ −10.5 (d, J = 22.9 Hz, 1P), −11.5 (d, J = 22.3 Hz, 1P), −23.2 (t, J = 22.3 Hz, 1P). HRMS [ESI-MS] calc. for C₁₀H₁₆N₆O₁₂P₃⁻, [M-H]⁻: m/z 505.0045, found m/z 505.0040.

2.2. Restriction and Modification Enzymes, Plasmids and E. coli Strains

Restriction endonucleases (REases), high-fidelity Phusion and Q5 DNA polymerases were provided by New England Biolabs (NEB). Restriction digests were carried out in the recommended restriction buffers at the appropriate temperatures: buffer 1.1 (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl₂, 100 µg/mL BSA or recombinant albumin, pH 7.0 at 25 °C); 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 100 µg/mL BSA or recombinant albumin, pH 7.9 at 25 °C); 3.1 (100 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 100 µg/mL BSA or recombinant albumin, pH 7.9 at 25 °C) or CutSmart buffer (50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 µg/mL BSA or recombinant albumin, pH 7.9 at 25 °C). Plasmids pBR322, pUC19, 2 log DNA ladder (1 kb plus), and phage λ DNA were also from NEB. The T7 expression vector pET21b with C-terminal 6× His tag was originally purchased from Novagen (NdeI-XhoI). E. coli competent cells of the following strains were produced in-house at NEB: NEB Turbo (Mrr⁺, E. coli K strain), C2925 (Mrr⁺, Dam⁻, Dcm⁻, McrA⁻, McrB⁻), NEB 10β (RecA⁻), C2683 (Mrr⁻, McrBC⁻, K strain), T7 shuffle (C3026, K strain), Nico (λDE3) (C2529, B strain), NEB Express (C2523, B strain). T7 Express (C2566) was used as a host for T7 expression vectors. pACYC-T7 was a derivative of pACYC184 (Cm^R) that contains phage T7 promoter and T7 transcription terminator, which is compatible with pET21b. Synthetic gene blocks were purchased from IDT. Plasmid mini-prep kit and PCR DNA clean-up kits were provided by NEB.

2.3. Cell Culture Conditions

E. coli cells containing pET21 vector (ColE1 replication origin) with or without gene insert were cultured overnight at 25–37 °C in LB supplemented with Amp (100 µg/mL). E. coli cells carrying pACYC-T7 (p15A replication origin) with or without gene insert were cultured overnight at 25–37 °C in LB supplemented with Cm (33 µg/mL).

2.4. dZ Production in E. coli Cells

For dZ production in E. coli cells, cells with plasmid carrying dZ synthetic pathway were cultured in LB plus antibiotics to mid-log phase, cells were cooled down (no shaking) at room temperature for 30 min, then IPTG (0.3–0.5 mM) was added to the cells. Cells were cultured in a shaker at 25 °C overnight (15–16 h). Cell pellets were obtained in a centrifuge, and plasmid DNA was extracted by using a mini-prep plasmid kit (NEB #T1110S).

2.5. LC-MS Analysis

LC-MS analysis was performed using an Agilent 1290 Infinity II UHPLC equipped with G7117A Diode Array Detector and 6135XT MS Detector (Agilent Technologies, Santa Clara, CA, USA), on a Waters XSelect HSS T3 XP column (2.1 × 100 mm, 2.5 µm) (Waters Corporation, Milford, MA, USA) with the gradient mobile phase consisting of methanol and 10 mM ammonium acetate buffer (pH 4.5). The relative abundance of each nucleoside was determined by the integration of each peak at 260 nm or its respective UV absorption maxima.

3. Results

dZTP was synthesized following a published procedure with minor modifications and increased scale [13,14], which afforded dZTP at ~28% yield and ~98% purity. Figure 1 shows the reaction scheme for dZTP synthesis and mass confirmation of the synthetic product by HRMS.

3.1. Restriction Digestion of dZ-Modified PCR DNA

We used two sets of primers and Q5 DNA polymerase to amplify dZ-modified PCR DNA (3 and 4 kb) from pBR322. dZ-containing DNA substrates were utilized for restriction analysis. LC-MS base composition analysis indicated 98.6% of dZ incorporation in the DNA substrate (Figure 2).

REases with one to six dZ within the recognition sequences were selected for restriction digest. Figure 3 shows 18 examples of digests of dZ-modified or unmodified (dA) PCR DNA. Four types of digestion were detected: fully resistant (R), mostly resistant (MR), partially resistant (PR, less than 50% of the DNA is resistant), and sensitive (S, complete or nearly complete digestion). Among the 18 digests, four enzymes (ApoI, BstYI, BsaI-HF v2, and SalI) can fully or nearly completely digest dZ-modified DNA. Comparing the restriction patterns for dZ (top) and dA (bottom) substrates, the modified substrate is resistant or partially resistant to 15 restriction enzymes (Figure 3A). REases in Figure 3B, can all completely digest unmodified DNA. Of the 147 REases examined, dZ-containing DNA was resistant or partially resistant to about 53%, while 47% were able to completely digest the modified DNA.

Table 1. Type II restriction of dZ-containing PCR DNA.

Type II Restriction Level	Number of REases	Percentage Among 147 REases
Fully resistant (R) + mostly resistant (MR)	49	33.3%
Partially resistant	29	19.7%
R/MR + PR	78	53.1%
Sensitive (non-resistant)	69	46.9%
Total REases tested	147

summarizes the restriction results. Supplementary Materials Table S1 lists individual restriction results. Compared to other hypermodified phage DNA, such as glc-5hmC-modified T4 genome, which is resistant to over 90 REases with CG recognition sequences, the dZ-modified DNA displays moderate resistance to Type II restriction. Only one ATP-dependent Type III restriction enzyme, EcoP15I (CAGCAG N25/27), was tested on the dZ substrate, and it showed no cleavage activity. We also tested one Type IV restriction system (Mrr) in vivo by plasmid transformation because its restriction activity had been shown on 6mA- and 5mC-modified DNA (see below).

Interestingly, some REases with similar recognition sequences had different outcomes on the restriction of dZ-modified DNA. For example, EarI (CTCTTC N1/N4) was resistant to dZ DNA, but SapI and BspQI (GCTCTTCN1/N4) were partially resistant. EcoO109I (RGGNCCY) was sensitive to dZ DNA, but PpuMI (RGGWCCY) was resistant. A single dZ in GGWCC blocked AvaII digestion; however, GCWGC was sensitive to ApeKI restriction in dZ-modified DNA. Of note, a limited number of Type IIS REases (enzyme cleavage downstream/outside of recognition sequences) were resistant to dZ modifications, including BbsI, BciVI, BfuAI, BpmI, BsgI, BsmI, and EcoP15I. This property may be utilized in recombinant DNA techniques, in which restriction sites in the primer with dZ modification are blocked, but internal sites in PCR DNA in the absence of modifications can be cut, a strategy similar to methylated or phosphorothioated sites in the primers. We have not tested the resistance of hemimodified PCR DNA substrates with dZ modifications.

3.2. Incorporation of dZ Base in E. coli

It has been shown that the dZ biosynthesis pathway can be expressed in E. coli to generate partially dZ-modified plasmid and genomic DNA [12]. The level of dZ incorporation can reach over 20% in plasmid DNA carrying the dZ biosynthesis genes under IPTG induction. Our goal was to improve dZ incorporation by co-expressing three genes from Salmonella phage for dZ biosynthesis and a fourth gene encoding phage DNA polymerase. In a preliminary experiment, we examined the dZ level in expression plasmids and found that the three-gene (pET21b-purZ-mazZ-datZ) and four-gene systems (pET21b-purZ-mazZ-datZ-dpoZ) generated 19% and 30% of dZ (dZ over dZ + dA), respectively, under IPTG induction overnight at 18 °C. This dZ incorporation level is slightly higher than that of the reported expression of three genes in E. coli from cyanophage S-2L, which was in the range of 7–16% [12]. To further increase the dZ level, we grew the T7 expression strain carrying four genes to mid-log phase and performed IPTG induction at 25 °C overnight.

Table 2. dZ/(dZ + dA) level in four-gene plasmids (samples 1–12) by LC-MS analysis. Negative control pBR322 (sample 13 contains 0% dZ). Two data points were obtained for all 13 samples.

	dZ/(dZ + dA) × 100% Measure 1	dZ/(dZ + dA) × 100% Measure 2
Sample 1	29%	30%
Sample 2	38%	38%
Sample 3	42%	42%
Sample 4	14%	14%
Sample 5	33%	33%
Sample 6	35%	35%
Sample 7	40%	40%
Sample 8	35%	35%
Sample 9	44%	44%
Sample 10	40%	40%
Sample 11	30%	30%
Sample 12	29%	29%
Sample 13 (vector)	0%	0%

shows that dZ reached as high as 44% in sample #9. Among the 12 plasmid samples tested, 11 exhibited dZ levels between 29% and 44%. Representative base composition analysis by LC-MS is shown in Figure 4A,B. In principle, the host genomic DNA should also contain a high level of dZ, but we found difficult to accurately determine its percentage due to contaminating plasmid in genomic DNA preparation using commercially available kits for purification. CsCl₂ gradient separation of plasmid DNA from genomic DNA may offer a route to obtaining cleaner data.

We also explored the expression of a six-phage gene system in the same host by adding phage DNA helicase-dpoZ-SSB in a compatible plasmid, pACYCT7. Two plasmids, pET21b-purZ-mazZ-datZ and pACYCT7-DNA helicase-dpoZ-ssb, were co-transformed into a T7 expression strain C2566. After IPTG induction at 25 °C overnight, the dZ level reached 47% to 61%, which is on average higher than that of the four-gene plasmid system, although the T7 expression host carrying six phage genes grew slowly and reached a lower cell density after overnight culture. To see whether the dZ level was reproducible, we repeated the experiment by IPTG induction of the two expression plasmids in the same host (

Table 3. Individual measurement of dZ level in expression plasmids with 3–6 genes involved in dZ biosynthesis. (1). Three-gene plasmid: pET21b-purZ-mazZ-datZ, +IPTG (samples 7–9), no IPTG (samples 14–16). (2). Four-gene plasmid: pET21b-purZ-mazZ-datZ-dpoZ, + IPTG (samples 1–6). (3). Six-gene plasmids: pET21b-purZ-mazZ-datZ and pACYCT7-DNA helicase-dpoZ-ssb, +IPTG (samples 10–12).

Transformation	dC	dG	dT	dZ	dA	dZ/(dZ+dA) × 100%	dZ/(dZ+dA) × 100% Average
PurZ-MazZ-DatZ-DNA pol #1	1.036	1.000	0.931	0.319	0.569	35.91%	39.8%
PurZ-MazZ-DatZ-DNA pol #2	1.042	1.000	0.931	0.336	0.544	38.18%
PurZ-MazZ-DatZ-DNA pol #3	1.048	1.000	0.933	0.331	0.553	37.39%
PurZ-MazZ-DatZ-DNA pol #4	1.051	1.000	0.951	0.282	0.601	31.96%
PurZ-MazZ-DatZ-DNA pol #5	1.027	1.000	0.908	0.403	0.469	46.16%
PurZ-MazZ-DatZ-DNA pol #6	1.049	1.000	0.907	0.427	0.443	49.06%
PurZ-MazZ-DatZ+IPTG #1	1.024	1.000	1.008	0.068	0.879	7.22%	9.0%
PurZ-MazZ-DatZ+IPTG #2	1.029	1.000	1.009	0.100	0.846	10.55%
PurZ-MazZ-DatZ+IPTG #3	1.024	1.000	1.011	0.087	0.862	9.15%
PurZ-MazZ-DatZ;Helicase-DNA pol-SSB #1	1.034	1.000	0.933	0.558	0.357	60.99%	50.0%
PurZ-MazZ-DatZ;Helicase-DNA pol-SSB #2	1.030	1.000	0.938	0.382	0.508	42.90%
PurZ-MazZ-DatZ;Helicase-DNA pol-SSB #3	1.036	1.000	0.934	0.414	0.482	46.20%
PurZ-MazZ-DatZ. No IPTG #1	1.021	1.000	0.925	0.234	0.637	26.87%	27.5%
PurZ-MazZ-DatZ. No IPTG #2	1.013	1.000	0.917	0.243	0.629	27.87%
PurZ-MazZ-DatZ. No IPTG #3	1.013	1.000	0.919	0.242	0.629	27.81%
pBR322 vector	0.998	1.000	0.890	0.000	0.818	0.00%	0%

). Plasmids extracted from C2566 cells [pET21b-purZ-mazZ-datZ and pACYCT7- DNA helicase-dpoZ-ssb] gave rise to the best dZ incorporation at 50% (average of three samples/plasmids) (Figure 5, Supplementary Materials Figure S1). The four-gene plasmid yielded dZ levels at nearly 40% (average of six samples/plasmids). Surprisingly, the non-induced cells transformed with the three-gene plasmid system generated a higher dZ level than that of the IPTG-induced cells (27% vs. 9%). The underlying cause of this unexpected result is not yet understood.

3.3. Transfer of dZ-Modified Plasmid DNA into E. coli Lab Strains by Transformation

When modified DNA enters E. coli cells, it may encounter modification-dependent restriction systems such as McrBC (RglBC) [3,15], Mrr [16], and McrA [15], GmrSD [17], SauUSI [18], DpnI [19] and winged-helix fusion endonucleases or other EVE-HNH domain-containing endonucleases (reviewed in [20]). Therefore, in this work we examined the transformation efficiency of dZ-modified pUC19 DNA into commonly used E. coli lab strains, particularly Mrr⁺ strains, that are involved in the restriction of modified adenine in DNA [16]. A small reduction in transformation efficiency was observed in Mrr⁺ strains with the dZ-containing plasmid.

To examine the transformation efficiency of a dZ-modified plasmid into commonly used E. coli lab strains, we designed two sets of primers to amplify pUC19 by inverse PCR. The PCR DNA has overlapping sequences at the priming sites and anneals to form circular DNA, which can be transferred into E. coli competent cells without ligation. The dZ-modified pUC19 and regular pUC19 inverse PCR DNA were transferred into E. coli lab strains by transformation using 20 ng DNA: NEB Turbo (Mrr⁺, E. coli K strain), C2925 (Mrr⁺, Dam⁻, Dcm⁻, McrA⁻, McrB⁻), NEB 10β (Mrr⁻, RecA⁻), C2683 (Mrr⁻, McrBC⁻, K strain), T7 shuffle (C3026, K strain), Nico (λDE3) (C2529, B strain), NEB Express (C2523, B strain). A less than two-fold difference was observed in transformation efficiency when the dZ-modified pUC19 PCR DNA was transferred into the above strains compared to unmodified inverse PCR DNA, with the exception of two strains, NEB Turbo and C2925. In the NEB Turbo and C2925 strains, there was a reduction of 46- to 63-fold in transformation efficiency using dZ-modified DNA (average of two experiments). We inferred that Mrr endonuclease, which restricts modified adenosine (N6mA), is likely implicated in the reduced PCR DNA transformation, although other host genetic backgrounds may also be involved. Further study is needed to pinpoint the molecular basis of the reduced transformation. Typical RE systems restrict foreign DNA or phage DNA at 10³- to 10⁶-fold. Thus, a ~50-fold-reduced transformation into the Mrr⁺ strain is not a significantly large restriction. For cloning and working with dZ-modified DNA, it may be advisable to use Mrr-deficient E. coli strains: NEB 10β, C2683 (Mrr⁻, McrBC⁻), T7 shuffle (C3026), Nico (λDE3) (C2529), NEB Express (C2523), and C2566 (T7 Express). The dZ-modified pUC19 DNA (or modified pET21b-purZ-mazZ-datZ-dnpZ) can be used to transform WT E. coli strains and screen for modification-dependent restriction systems, specifically towards dZ DNA if such dZ-dependent REases exist in WT host strains. In the biological arms race between host and phages, coliphages such as T4 utilize DNA base hypermodifications to counteract host-encoded Type II restriction systems [6]. Subsequently, E. coli hosts evolve modification-dependent REases such as GmrSD and EcoO157SI to restrict modified T4 genomes with glc-5hmC (glucosylated-5hmC) [17], or McrBC and McrA to restrict modified cytosines in T4gt [15].

3.4. Exonuclease Digestion of dZ-Modified PCR DNA

If incorporation of dZ into DNA reduces susceptibility to Type II restriction, an important question is whether such a modification also confers resistance to exonuclease digestion. dZ and dA PCR DNA substrates were treated with the exonucleases RecBCD (supplemented with ATP), E. coli exonuclease III (exo III), phage T5 exonuclease (T5 exo), phage T7 exonuclease (T7 exo), E. coli exonuclease VIII (exo VIII, truncated), phage λ exonuclease (λ exo), and Bal31 nuclease. There appeared to be very minor differences in exonuclease digestions of dZ-modified PCR DNA in 20 min digestion compared to the digestion of regular PCR substrate. Bal31 exonuclease digestions were inconclusive due to the complete degradation of both dZ-modified and unmodified PCR DNA (1 U incubated with 0.35 μg DNA. As expected, MluCI (AATT) cleaved the unmodified PCR DNA, while most of the dZ-modified PCR DNA was not digested. To further confirm the digestion of dZ-modified DNA by RecBCD and T5 exonucleases, we performed a time-course reaction ranging from 1 to 30 min. The results are shown in Figure 6. There were minimal differences in RecBCD-mediated digestion of dZ-modified and unmodified PCR DNA over the 1–30 min incubation period. A minor difference was observed in the T5 exonuclease digestion of dZ-modified DNA. After 30 min, the dZ end products were slightly larger (0.5–1.5 kb) than the end products of dA PCR DNA (0.5–0.7 kb), suggesting that dZ-modified DNA slowed down T5 exonuclease degradation. A reduced digestion rate of dZ-modified DNA by certain exonucleases may be a desired feature for gene therapy and DNA vaccine applications to prolong the half-life of target genes.

4. Discussion

In this work, we demonstrated that dZ-modified PCR DNA is resistant or partially resistant to 53% of the REases examined, further supporting the notion that the main biological function of dZ modification is fighting against host-encoded restriction systems. A critical feature of dZ-containing phages is that phage-encoded DNA polymerase (PolI family) or primase–polymerase fusion (Pri-pol family) can efficiently replicate phage DNA from a nucleotide pool with dZTP [21]. We took advantage of this by co-expressing the dZ biosynthesis genes purZ, mazZ, and datZ with the Salmonella DNA polymerase gene dpoZ in E. coli to achieved 29% to 40% dZ incorporation in plasmids. We also showed that dZ-containing PCR DNA can reduce the rate of T5 exonuclease degradation based on a time-course study. Interestingly, dZ DNA can be degraded as well as dA DNA by the ATP-dependent exonuclease RecBCD. It has been previously shown that hypermodified XP12, SP-15, PBS1 phage DNAs can also slow down degradation by the non-specific endonuclease DNase I [22].

Expression of the plasmid containing Salmonella three-gene system (pET21b-purZ-mazZ-datZ) was shown to enable incorporation of dZ at ~20%, which is higher than the three-gene cluster cloned from cyanophage S-2L (7–14% dZ incorporation) [12]. One possible explanation is that Salmonella phage genes are more efficiently expressed in E. coli, likely because the gene blocks were synthesized using the native Salmonella phage DNA sequences. We have not examined individual expression of each of these three genes.

Mrr endonuclease is a Type IV REase in E. coli that restricts 6mA- and 5mC-modified plasmid and phage DNA. It was discovered during the cloning of foreign methylase genes into E. coli [23]. The other two Type IV REases involved in restriction of modified adenosines are winged-helix domain fusion endonucleases (e.g., DpnI and HhiV4I) and possibly Yth domain-containing fusion endonucleases [24,25]. We have seen previously that DpnI and HhivV4I endonucleases do not digest dZ-containing PCR DNA (unpublished results). Here, we report that when dZ-containing pUC19 PCR DNA was transferred into Mrr⁺ E. coli host, a small reduction (~50-fold) in transformation efficiency was observed. It is not clear whether this reduction is due to the transcription-coupled nucleotide-excision repair (TC-NER) in the host or the result of true restriction by the Mrr endonuclease. Further experiments are needed to answer this question. Regardless of restriction or DNA repair-based nicking, in applications involving dZ-modified DNA, it may be prudent to consider using Mrr-deficient E. coli strains.

This work has a general interest for molecular biologists working on dZ DNA modification and restriction systems. It provides a foundation for future research on screening dZ-dependent Type IV restriction systems. The results presented here may have implications in gene therapy utilizing dZ-modified DNA, provided that human RNA polymerase variants can efficiently perform transcription from a dZ-modified template.