Ribonucleoside triphosphates promote T7 DNA replication and the lysis of T7-Infected Escherichia coli

Biochimie 167 (2019) 25e33

Contents lists available at ScienceDirect

Biochimie journal homepage: www.elsevier.com/locate/biochi

Research paper

Ribonucleoside triphosphates promote T7 DNA replication and the lysis of T7-Infected Escherichia coli Zhenyu Zou a, Wendi Xu b, Chenyang Mi a, Ying Xu a, Ke Du a, Bianbian Li a, Yang Ye c, Yihui Ling d, Huidong Zhang a, * a Key Laboratory of Environment and Female Reproductive Health, West China School of Public Health & West China Fourth Hospital, Sichuan University, Chengdu, 610041, China b College of Biological Sciences and Engineering, North Minzu University, Yinchuan, Ningxia, 750021, China c Department of Obstetrics and Gynecology, Sun Yat-sen Memorial Hospital, Yanjiang West Road 107, Guangzhou, Guangdong, 510120, China d Institute for Chemical Carcinogenesis, Guangzhou Medical University, Xinzao, Panyu District, Guangzhou, 510000, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 April 2019 Accepted 1 September 2019 Available online 4 September 2019

rNTPs are structurally similar to dNTPs, but their concentrations are much higher than those of dNTPs in cells. rNTPs in solutions or rNMP at the primer terminus or embedded in template always inhibit or block DNA replication, due to the reduced Mg2þ apparent concentration, competition of rNTPs with dNTPs, and the extra repulsive interaction of rNTP or rNMP with polymerase active site. In this work, unexpectedly, we found rNTPs can promote T7 DNA replication with the maximal promotion at rNTPs/dNTPs concentration ratio of 20. This promotion was not due to the optimized Mg2þ apparent concentration or the direct incorporation of extra rNMPs into DNA. This promotion was dependent on the concentrations and types of rNTPs. Kinetic analysis showed that this promotion was originated from the increased fraction of polymerase-DNA productive complex and the accelerated DNA polymerization. Further evidence showed that more polymerase-DNA complex was formed and their binding affinity was also enhanced in the presence of extra rNTPs. Moreover, this promotion in T7 DNA replication also accelerated the lysis of T7infected host Escherichia coli. This work discovered that rNTPs could promote DNA replication, completely different from the traditional concept that rNTPs always inhibit DNA replication. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Keywords: Bacteriophage T7 DNA polymerase rNTP and rNMP dNTP incorporation DNA replication Lysis of Escherichia coli

1. Introduction DNA replication in cells is performed in a pool containing four dNTPs and four rNTPs. rNTPs are structurally similar to their corresponding dNTPs except for an extra hydroxyl group at the sugar ring, but their cellular concentrations are one to six orders of magnitude higher than those of dNTPs, depending on cell types and cell cycle stages [1]. The concentration ratios of rNTP/dNTP range from 1.8 to 20 in E. coli, 36 to 188 in budding yeast, and 10 to 133 in human normal cell (Table S1). Most DNA polymerases (Pols) use a bulky amino acid residue called the ‘steric gate’ at the entrance of their active sites to select dNTPs and discriminate against rNTPs [2,3]. Escherichia coli (E. coli) DNA Pol I (KF exo), bacteriophage T7 DNA polymerase (Pol T7),

* Corresponding author. E-mail address: [email protected] (H. Zhang).

Sulfolobus solfataricus P2 DNA Pol Dpo4, and human DNA Pol ɩ select dNTPs at least three orders of magnitude more preferentially than rNTPs (Table S2). The steric gate amino acid residues are generally Glu for A-family DNA polymerases and Tyr or Phe for B-, X-, Y-, or RT-families [4]. Reducing the steric gate residue size generally increases rNTP incorporation capability [4,5]. Till now, it has been reported that rNTPs inhibit DNA replication by B-family eukaryotic DNA Pols d and ε [6]. The addition of rNTP that can pair with the template base retards primer extension by DNA Pol m [7]. Crystal structures of the complex of hPol h, DNA, and incoming rNTP show that the distance between the primer terminus and the Pa of rNTP is increased, which inhibits DNA polymerization compared with that using dNTP [1]. Moreover, the ternary complex structure of Pol b with an undamaged template and an incoming rCTP shows the repulsive interaction between the backbone carbonyl of Tyr271 and the 20 -OH group of rCTP, resulting in a disturbed active site geometry [8]. In E. coli, increasing concentrations of rNTPs reduces the primer extension by DNA Pol III complex

https://doi.org/10.1016/j.biochi.2019.09.002 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

26

Z. Zou et al. / Biochimie 167 (2019) 25e33

and also decreases the progression rate of the leading-strand DNA synthesis by E. coli DNA replisome [6]. This inhibitory effect is originated from the competition of rNTPs with dNTPs at the active sites of DNA polymerase. Additionally, rNTPs can also chelate free Mg2þ ion and reduce its apparent concentration in solution. However, rNTPs are inevitably incorporated into DNA to form rNMPs by various DNA polymerases, due to the imperfect exclusion of rNTPs from the polymerase active site and high rNTPs/dNTPs ratio in cells [5,6,9]. The frequencies of rNTP incorporation were estimated at 104-105 or at a lower level for E. coli Pol I KF exo, Pol T7, Dpo4, and hPol ɩ (Table S2). These rNMPs embedded in template impede, retard, or block DNA replication by bacteriophage RB69 Pol [9], yeast Pols a, d, and ε [10], T7 or E. coli DNA replisome [6,11]. A structural study of RB69 Pol reveals that rNMP in template alters sugar pucker and the positions of two conserved crucial amino acids at RB69 Pol active site, thus reducing DNA replication efficiency [12]. rNMP at primer terminus also reduces primer extension efficiency compared with that from dNMP, as observed for hPols g, ε, and m [4,13]. Till now, all the reported results show that rNTPs in solution or rNMP at primer terminus or embedded in template inhibits or even blocks DNA replication. In this work, unexpectedly, we found that rNTPs/dNTPs at appropriate concentration ratios can promote T7 DNA replication. This promotion was because more polymeraseDNA productive complex was formed and their binding affinity was increased in the presence of appropriate concentrations of rNTPs. This promotion also accelerates the lysis of T7-infected E. coli. This study reveals novel functions of rNTPs in the promotion of DNA replication, completely different from the traditional concept that rNTPs always inhibit DNA replication. 2. Material and methods 2.1. Materials T4 polynucleotide kinase, rNTPs, dNTPs, KF exo, RNase H1, and RNase H2 were purchased from New England Biolabs (Beverly, MA). [g-32P] ATP (specific activity 3  103 Ci mmol1) was from PerkinElmer Life Sciences (Boston, MA). Oligodeoxynucleotides in Table S3 were synthesized and purified by HPLC (Takara Bio, Kyoto, Japan). Recombinant T7 DNA polymerase (gp5) or exonucleasedeficient polymerase (gp5 exo), E. coli thioredoxin (trx), Dpo4, hPol i1-445, and Gp90 exo were expressed in E. coli and purified as previously described [14e17]. For simplicity, we denote gp5 exo/ trx complex as Pol T7, gp5/trx as Pol T7, and hPol i1-445 as hPol i throughout this manuscript. Bacteriophage T7 was provided from Charles C. Richardson at Harvard Medical School. All other reagents were of the highest quality commercially available. 2.2. Primer extension in the presence of dNTPs and/or rNTPs Primer extension by Pol T7 was performed by mixing 15 nM Pol T7, 20 nM 32P-labeled 27-mer/62a-mer primer/template DNA substrate, 0.2 mM each of four dNTPs, and 0, 1, 2, 4, 8, 12, or 16 mM each of four rNTPs in buffer A (40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, and 50 mM potassium glutamate) at 37  C for 0.5, 1, or 3 min Gp5 exo was incubated with 20-fold molar excessive E. coli trx at 4  C for 5 min to form Pol T7 complex [14]. After termination of reactions by the addition of EDTA quench solution, the samples were denatured and separated on a 20% polyacrylamide (w/v)/7 M urea gel. Products were visualized using a phosphorimaging screen and quantified by Quantity One™ software [18,19]. Primer extension by Pol T7 was also performed under different conditions: 5 nM Pol T7 and 20 nM 32P-labeled 27-mer/62a-mer,

in the presence of rNTPs alone, in the presence of one, two, three, or four types of rNTPs at a fixed concentration, using Pol T7 instead of Pol T7, using Pol T7 purified from another batch, using varying concentrations of Pol T7, or using another 24-mer/36-mer DNA substrate with random sequences (sequences in Table S3). Primer extension was also performed with varying concentrations of Mg2þ in Buffer C (40 mM Tris-HCl (pH 7.5), 10 mM DTT, and 50 mM potassium glutamate). Steady-state kinetic analysis of single-nucleotide incorporation opposite G by Pol T7 was performed by mixing Pol T7, 32Plabeled 27-mer/62a-mer DNA substrate, and a varying concentration of single dCTP or rNTP in buffer A, as described previously [20e22]. The kcat and Km values were obtained. 2.3. DNA cleavage assays by RNase H1 or RNase H2 Cleavage assays [23] were performed by mixing 20 nM 62bmer/62c-mer dsDNA with 2 units of RNase H1 in buffer D (10 mM Tris-HCl pH 7.5, 35 mM KCl, 1 mM DTT, 4 mM MgCl2 and 5% Glycerol) or mixing 20 nM 62b-mer/62c-mer dsDNA with 2 units RNase H2 in buffer E (20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 2 mM MgSO4) at 37  C for 15 min. The 62b-mer strand was labeled with 32P at its 50 -terminus and contained dA or rA for RNase H2 assays or contained dA, r(AU), r(AUC), or r(AUCG) for RNase H1 assays. After termination of reactions by EDTA solution, the samples were denatured and the cleaved products were separated on a 20% polyacrylamide (w/v)/7 M urea gel and visualized [24]. Primer was extended in the presence of 5 nM Pol T7, 20 nM 32 P-labeled 27-mer/62a-mer, 0.2 mM each of four dNTPs, and 4 mM each of four rNTPs in buffer A at 37  C for 3 min. Afterward, the reaction sample was heated at 95  C for 10 min to deactivate polymerase, slowly cooled down to re-anneal DNA, and passed through a Bio-Spin 6 column to purify DNA. The extended products were subsequently used as substrates in cleavage assays by RNase H1 or H2. 2.4. Kinetic analysis of primer extension in the presence of extra rNTPs Rapid quench experiments were performed using a model RQF-3 KinTek Quench Flow Apparatus (KinTek Corp, Austin, TX, USA) with 40 mM Tris-HCl buffer (pH 7.5) in both drive syringes and 0.5 M EDTA solution in the middle quench syringe [17,19]. Primer extension was performed by rapidly mixing 40 nM Pol T7, 20 nM 32Plabeled 27-mer/62a-mer DNA, and 0.2 mM each of four dNTPs in the absence or presence of 4 mM each of rNTPs in buffer A at 37  C. After incubation at varying time, the reactions were quenched, and the products were separated, visualized, and quantified [19,25,26]. The 62-mer product concentration and time were fit to Eq. (1) [11,26]. y ¼ A (1e-k t)

(1)

where y is the 62-mer product concentration, nM; A is the fraction of productive complex for full-length primer extension, nM; k is the average primer extension rate, min1; and t is time, min. 2.5. The formation of Pol T7 and DNA complex determined by gel mobility shift assay Gel mobility shift assay [27] was performed by mixing 5 nM Pol T7 and 20 nM 32P-labeled 27-mer/62a-mer with 0.2 mM each of four dNTPs and 400 nM unlabelled 27-mer/62a-mer in the absence or presence of 4 mM each of four rNTPs in buffer A at 37  C. A Cdd at

Z. Zou et al. / Biochimie 167 (2019) 25e33

the primer terminus blocked DNA polymerization. After incubation at 37  C for 10 min, the mixture was loaded on an 8% TBE nondenaturing gel and electrophoresed at 75 V at 4  C for 1 h in 0.5 x TBE buffer. The gel was then dried for autoradiography. 2.6. Binding affinity of Pol T7 to DNA determined by Surface Plasmon Resonance (SPR) Binding of Pol T7 to 30-mer/62a-mer primer/template was determined by SPR using a Biacore 3000 instrument (Uppsala, Sweden) [11,27,28]. DNA substrate (300 response units, RU) was immobilized to a streptavidin (SA) chip via a biotin at the 50 primer terminus. A Cdd at the 30 end of primer blocked DNA polymerization. Biotin was used in control to compensate for the background. Pol T7 (20 nMe200 nM) in buffer A containing 0.2 mM each of four dNTPs and 0 or 4 mM each of four rNTPs was flowed over the chip at 10 mL/min at room temperature to study the binding affinity of Pol T7 to DNA. To obtain the dissociation constants Kd, the binding signal and polymerase concentration were fit to Eq. (2). The chip surface was regenerated by injection of 150 mL of 1 M NaCl at a flow rate of 100 mL/min. Y ¼ B  RUmax/(B þ Kd)

(2)

where Y is the response signal of binding, RU; B is Pol T7 concentration, nM; RUmax is the maximal binding amount, RU; and Kd is the dissociation constant, nM. All experiments were replicated thrice, and standard errors were derived using GraphPad Prism Version 6.0. 2.7. Growth curves of E. coli DH5a or T7-infected E. coli in LB media containing extra four rNTPs E. coli DH5a (OD600 of 0.9, 40 mL) were incubated with 160 mL LB

27

medium containing 0, 1, 2, or 4 mM each of four rNTPs (final concentration) in a 96-well plate at 37  C [29,30]. The same 50 mL E. coli DH5a (OD600 of 0.9) and 60 mL bacteriophage T7 (6.5  106 pfu) in LB medium containing 0, 1, 2, or 4 mM each of four rNTPs (final concentration) were incubated in a 96-well plate at 37  C. OD600 measurements were taken every 5 min by a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA) and E. coli growth curves were recorded.

3. Results 3.1. rNTPs promoted the full-length primer extension by T7 DNA polymerase Full-length primer extension by Pol T7 was examined in the presence of 0.2 mM each of four dNTPs and increasing concentrations (0e16 mM) of each of four rNTPs (Fig. 1). Primer extension was first promoted and reached the maximum at the rNTPs/dNTPs concentration ratio of 20 (4 mM each of four rNTPs) and was then gradually inhibited (Fig. 1A). The 62-mer product concentrations after reaction for 1 min were quantified and plotted against rNTPs/ dNTPs concentration ratios (Fig. 1B), clearly showing that rNTPs promoted this primer extension. Generally, the presence of extra rNTPs always inhibit DNA replication [6,31]. It was the first time that we found rNTPs could promote DNA replication at appropriate rNTPs/dNTPs ratio. The similar assays were also performed using other DNA polymerases (Fig. S1). Gp90 exo [16,17], E. coli KF exo, and Pol T7 are A-family DNA polymerases. Dpo4 and hPol ɩ are Y-family polymerases. Primer extension by Gp90 exo, Dpo4, and hPol ɩ was gradually inhibited with increasing rNTP concentrations (Figs. S1A, D, E). However, primer extension by KF exo and Pol T7 could be promoted at lower rNTP concentrations but was inhibited till blocked at higher rNTP concentrations (Figs. S1B and C). The

Fig. 1. Primer extension by Pol T7 was promoted in the presence of four rNTPs in an appropriate rNTPs/dNTPs ratio range. A, Extension assays were performed by mixing 15 nM Pol T7, 20 M32P-labeled 27-mer/62a-mer DNA, 0.2 mM each of four dNTPs, and 0, 1, 2, 4, 8, 12, or 16 mM each of four rNTPs in buffer A at 37  C for 0, 0.5, 1, or 3 min. The numbers on the left depict the substrate and product. Representative data from three independent experiments are shown. B, The 62-mer product concentrations after reaction for 1 min were quantified and plotted against rNTPs/dNTPs concentration ratios. The error bars represent the standard deviation of three independent experiments.

28

Z. Zou et al. / Biochimie 167 (2019) 25e33

maximal promotion for Pol T7 was also observed at the rNTPs/ dNTPs concentration ratio of 20 (Fig. S1H).

To further validate this promotion effect, primer extension was performed under different conditions. Primer extension by wildtype Pol T7 was also promoted in the presence of 20-fold molar excessive four rNTPs (Fig. 2A). However, this extension was completely inhibited in the presence of only four rNTPs. Similar promotion was also observed with increasing concentrations of Pol T7 (Fig. 2B) or Mg2þ (Fig. 2C), using newly purified Pol T7 (data not shown), or using another 24-mer/36-mer DNA substrate with random sequences (Fig. 2D). All these results further confirmed that rNTPs/dNTPs at appropriate concentration ratios could promote primer extension by Pol T7, and the maximal promotion was constant at rNTPs/dNTPs concentration ratio of 20.

increased with increasing Mg2þ concentration (0e30 mM) in a dose-dependent manner in the presence or absence of extra rNTPs (Fig. 3B). With further increasing Mg2þ concentration (50e100 mM), the extension products increased slowly or gradually decreased, a trend that was similar to that reported previously [32]. No maximal primer extension activity was observed below 10 mM Mg2þ, showing no optimal Mg2þ concentration for T7 DNA polymerization in this tested Mg2þ concentration range. Furthermore, primer extension was more efficiently in the presence of extra rNTPs in all the Mg2þ concentration range. As expectedly, Mg2þ apparent concentration should be decreased in the presence of rNTPs. If extension activity is indeed dependent on Mg2þ apparent concentration, DNA polymerization activity should be decreased with increasing rNTP concentration. In contrast, our experiments showed that the addition of rNTPs increased the primer extension activity in the range of Mg2þ concentrations. Therefore, this promotion by rNTPs was not due to the optimized Mg2þ apparent concentration in solution.

3.3. The promotion by rNTPs was not due to the optimized Mg2þ concentration

3.4. The promotion by rNTPs was not due to the direct incorporation of extra rNTPs into DNA

The reasons why DNA polymerization was promoted by rNTPs were investigated. An identical volume of rNTP aqueous solution was used in all the assays, eliminating the possibility that the solvent of rNTPs affected DNA polymerization. Another possibility is that rNTPs could chelate free Mg2þ ions and reduced its apparent concentration in solution, possibly reaching an “optimized Mg2þ concentration” for T7 DNA polymerization thus giving maximal extension activity. In all these assays, the identical 10 mM Mg2þ was used. To verify this possibility, the effects of Mg2þ concentrations on primer extension was investigated in a much wider range (0e100 mM) (Fig. 3A). The extension efficiency was monotonically

Another possibility is that rNTPs might be directly incorporated into DNA by T7 DNA polymerase and promotes DNA replication. To test this hypothesis, primer was extended in the presence of only the identical amount of rNTPs (i.e., no dNTPs). Primer was very weakly extended in the presence of 1 or 2 mM each of four rNTPs and was completely blocked in the presence of 4e16 mM each of four rNTPs (Fig. S2). Thus, rNTPs alone significantly inhibited primer extension. Possibly, rNTPs might be incorporated during DNA polymerization in the presence of four dNTPs. Incorporation of rNTPs into DNA forms rNMPs. RNase H2 or RNase H1 could efficiently remove

3.2. rNTPs promoted Pol T7- DNA polymerization under various conditions

Fig. 2. Primer extension by Pol T7 was promoted in the presence of four rNTPs under different conditions. A, Extension assays were performed by mixing 5 nM Pol T7 and 20 nM 32 P-labeled 27-mer/62a-mer DNA substrate in the presence of 0.2 mM each of four dNTPs, 0.2 mM each of four dNTPs and 4 mM each of four rNTPs, or 4 mM each of four rNTPs in buffer A at 37  C for 0.5 min. B, Assays were performed similarly to (A), except that 0, 5, 10, 20, or 40 nM Pol T7 was used. C, Assays were performed similarly to (A) except that 5 nM Pol T7 and 0, 2, 5, 10, or 20 mM Mg2þ were used. D, Assays were performed with conditions similar to (B), except for using 24-mer/36-mer substrate with random sequences. Yields of the full-length product were quantified in percentage and were plotted against rNTPs/dNTPs ratios. The numbers on the left depict the substrate and products. Representative data from three independent experiments are illustrated. The error bars represent the standard deviation of three independent experiments.

Z. Zou et al. / Biochimie 167 (2019) 25e33

29

Fig. 3. Primer extension by Pol T7 was gradually promoted with increasing Mg2þ concentrations. A, Extension assays were performed by mixing 5 nM Pol T7-, 20 nM 32P-labeled 27-mer/62a-mer DNA substrate, 0.2 mM each of four dNTPs, 0 or 4 mM each of four rNTPs, and 0e100 mM Mg2þ in buffer C at 37  C for 0.5 min. Representative data from three independent experiments are illustrated. B, The 62-mer product concentrations were quantified and plotted against Mg2þ concentrations. The error bars represent the standard deviation of three independent experiments.

a single rNMP or consecutive rNMPs embedded in DNA, respectively, and cleaves DNA into short fragments [23]. To test this hypothesis, primer was extended in the presence of dNTPs and 20fold molar excessive rNTPs, and the extension products were treated with RNase H1 or RNase H2. In control assays, both RNase H1 and H2 cannot cleave the natural dsDNA. RNase H2 could cleave dsDNA containing a single rNMP in one strand; and RNase H1 could cleave dsDNA containing two, three, or four consecutive rNMPs in one strand (Fig. 4A). After cleavage, short DNA fragments were separated and identified. In our assays, no obviously cleaved products were observed for Pol T7 extension products in the presence of dNTPs and extra rNTPs (Fig. 4B), confirming that rNTPs

could not be significantly incorporated into DNA. Thus, the promotion for Pol T7 DNA polymerization was not due to the direct incorporation of extra rNTPs into DNA. Furthermore, incorporation of a single rNTP by T7 DNA polymerase was also kinetically analyzed (Table 1). dCTP was preferentially incorporated opposite dG. All rNTP incorporations were four to six orders of magnitude less efficient than dCTP incorporation, mainly due to the increased Km values. These results further confirmed that rNTPs were very inefficiently incorporated into DNA and the promotion for Pol T7 DNA polymerization was not due to the direct incorporation of rNTPs into DNA.

Fig. 4. The full-length extension products in the presence of four rNTPs could not be cleaved by RNase H1 or RNase H2. A, Cleavage assays were performed by mixing 2 units of RNase H2 with 20 nM 62b-mer/62c-mer DNA containing dA or rA in buffer E, or mixing 2 units of RNase H1 with 20 nM 62-bp dsDNA containing dA, r(AU), r(AUC), or r(AUCG) in buffer D at 37  C for 15 min. B, Primer extension was performed with 5 nM Pol T7, 20 nM 32P-labeled 27-mer/62a-mer, 0.2 mM each of four dNTPs, and 4 mM each of four rNTPs in buffer A at 37  C for 3 min. The sample was heated, slowly cooled down, and passed through Bio-Spin 6 column. The extension products were then used as substrates in these cleavage assays by RNase H1 or H2. The numbers on the left depict the substrates. Representative data from three independent experiments are shown.

30

Z. Zou et al. / Biochimie 167 (2019) 25e33

Table 1 Steady-state kinetic parameters for single-nucleotide incorporation opposite G by Pol T7. Template

Nucleotide

kcat  103 (s1)

Km (mM)

kcat/Km (mM1 s1)

fa

dG

dCTP rCTP rATP rUTP rGTP

7 ± 0.2 14 ± 0.2 2 ± 0.3 5 ± 0.3 3 ± 0.3

(3.9 ± 0.4)  103 32 ± 1 190 ± 20 29 ± 13 48 ± 18

1.8 4.4  104 1.0  105 1.7  104 6.3  105

2.4  104 5.6  106 9.4  105 3.5  105

a

Misincorporation frequency is defined as f ¼ (kcat/Km)rNTP/(kcat/Km)dCTP.

3.5. Promotion for Pol T7 polymerization was dependent on the concentrations and types of rNTPs The concentrations of rNTPs affected the T7 primer extension. Then, the effects of rNTP types on T7 DNA polymerization were further investigated. The presence of four types of 4 mM rNTPs promoted primer extension by Pol T7, similar to our previous results; however, the presence of any type of 4 mM rNTP inhibited this primer extension (Fig. 5A). Furthermore, several types of rNTPs were added based on the template complementary sequence. G was located at the first incorporation position in template and the addition of 4 mM rCTP did not promote this primer extension (Fig. 5B). The addition of two (rCTP and rGTP) or three (rCTP, rGTP, and rATP) based on the template complementary sequence did also not promote this extension. The addition of all four types of rNTPs could promote this extension. Possibly, it might be the total rNTP concentration that promotes DNA polymerization. Then, each type of rNTP at high concentration (16 mM) was examined. The addition of any type of 16 mM rATP, rGTP, or rCTP, but not rUTP, could promote this primer extension (Fig. 5C). However, the addition of all four types of 16 mM rNTPs completely inhibited this extension. These results showed that this promotion for Pol T7 polymerization was dependent on both the concentrations and types of rNTPs. 3.6. rNTPs increased the fraction of polymerase-DNA productive complex and also accelerated DNA polymerization Primer extension can be simplified into two steps [11,26]: (1) DNA polymerase binds DNA substrate to form polymerase-DNA productive complex, and (2) this productive complex extends

primer to the full-length product. The kinetic parameters were estimated for primer extension in the absence or presence of 4 mM each of four rNTPs (Fig. 6A). Molar excessive polymerase relative to DNA substrate was used to ensure that all DNA substrate was associated with polymerase. T7 DNA polymerase has high processivity in primer extension with hundreds of nucleotides in one binding event [33]. The 62-mer extension product and time were fit to a single exponential Eq. (1). rNTPs increased the fraction of polymerase-DNA productive complex (A) and primer extension average rate (k1) (Fig. 6B). Therefore, rNTPs accelerated primer extension by increasing the fraction of productive complex and DNA polymerization rate.

3.7. More Pol T7 and DNA complex was formed in the presence of extra rNTPs The formation of Pol T7 and 32P-labeled 27-mer/62a-mer DNA complex in the absence or presence of four rNTPs was determined by gel mobility assays [27] (Fig. 7). A Cdd at the 30 terminus of primer blocked DNA polymerization. The addition of 20-fold molar excessive unlabelled 27-mer/62a-mer DNA could trap any polymerase that had dissociated from the labeled DNA. T7 polymerase and DNA complex migrated slowly relative to free DNA in a nondenaturing gel. These assays show that more polymerase and DNA complex was formed in the presence of 20-fold molar excessive rNTPs (Fig. 7), agreed with that rNTPs promoted the formation of polymerase-DNA productive complex (Fig. 6B). These results explained the promotion for T7 DNA polymerization in the presence of extra rNTPs.

Fig. 5. The promotion for Pol T7 primer extension was dependent on the concentrations and types of rNTPs. Extension assays were performed by mixing 5 nM Pol T7, 20 nM 32Plabeled 27-mer/62a-mer, 0.2 mM each of four dNTPs, and varied concentrations of rNTPs [A, 4 mM each individual rNTP; B, 4 mM one to four types of rNTPs; or C, 16 mM each individual rNTP] in buffer A at 37  C for 1 min. The numbers on the left depict the substrate and product. The template sequence and full-length extension percentages are shown at the top of these panels. Representative data from three independent experiments are shown.

Z. Zou et al. / Biochimie 167 (2019) 25e33

31

Fig. 6. The presence of rNTPs increased the fraction of polymerase-DNA productive complex and the average rate of primer extension. A, Primer extension assays were performed with 40 nM Pol T7, 20 nM 32P-labeled 27-mer/62a-mer, 0.2 mM each of four dNTPs, 0 or 4 mM each of four rNTPs in buffer A at 37  C. B, The full-length 62-mer product and time were fit to a single exponent Eq. (1) to estimate kinetic parameters (productive complex A and primer extension average rate k). The solid lines represent the fit curves. Representative data from three independent experiments are presented. The averages and standard errors were obtained from three independent assays by fitting using Prism.

Fig. 7. More Pol T7 and DNA complex was formed in the presence of four rNTPs. Gel mobility shift assays were performed by mixing 5 nM Pol T7, 20 nM 32P-labeled 27mer/62a-mer, 0.2 mM each of four dNTPs, and 400 nM unlabelled 27-mer/62a-mer trap DNA in the absence or presence of 4 mM each of four rNTPs in buffer A at 37  C. Representative data from three independent experiments are shown.

3.8. rNTPs enhanced the binding affinity between DNA polymerase and DNA Subsequently, the binding affinity between DNA polymerase and primer/template DNA substrate in the absence or presence of extra four rNTPs was determined by Surface Plasmon Resonance (SPR) [27,34]. The 30-mer/62a-mer DNA (300 RU) was immobilized onto a streptavidin chip via a biotin at the 5’ end of primer. A Cdd at the 30 terminus of primer blocked DNA polymerization. Varying concentrations of DNA polymerase were flowed over the chip to measure the binding between polymerase and DNA in the absence or presence of extra rNTPs (Fig. 8AeC). DNA polymerase bound to DNA to form a binary complex in the absence of dNTP or Mg2þ, giving a dissociation constant (Kd) of 268 nM (Fig. 8D). In the presence of dNTPs and Mg2þ, polymerase, DNA, and dNTP formed a ternary complex, and the binding affinity of Pol T7 to DNA was enhanced (Kd of 140 nM, Fig. 8E). This binding affinity was further enhanced in the presence of extra 20-fold molar excessive four rNTPs (Kd of 73 nM, Fig. 8F). These results showed that rNTPs enhanced the binding affinity between polymerase and DNA, promoting the formation of more polymerase-DNA productive complex. 3.9. rNTPs promoted the lysis of T7-infected E. coli The effects of rNTPs on the growth of E. coli or T7-infected E. coli were investigated. E. coli growth was monitored at 600 nm, at

which rNTPs exhibited no absorbance. rNTPs can cross cell membrane through passive diffusion or facilitated uptake [35e37]. The addition of 1 mM each of four rNTPs (final concentration) in LB media accelerated E. coli growth compared with that in the absence of rNTPs (Fig. 9A), indicating that rNTPs could penetrate into E. coli and accelerate its growth. However, further increasing rNTP concentration to 2 mM and 4 mM gradually inhibited E. coli growth. In E. coli, DNA Pol III complex takes the major roles in DNA replication. It has reported that E. coli DNA replication by Pol III complex or by E. coli DNA replisome was gradually inhibited with increasing rNTP concentration in a dose-dependent manner [6]. Therefore, the inhibition in E. coli growth at higher rNTP concentrations was possibly originated from the inhibited E. coli DNA replication due to rNTPs. T7 is a lytic phage of E. coli. E. coli growth was inhibited after addition of T7, showing relatively flat growth curves compared with that without T7 (Fig. 9B). The rNTPs/dNTPs concentration ratios in E. coli were in the ranged from 1.8 to 20 (Table S1). Our in vitro results have shown that rNTPs could promote T7 DNA replication if the rNTPs/dNTPs concentration ratios were less than 20. Therefore, the addition of extra rNTPs into E. coli should promote T7 DNA replication, thus increasing T7 propagation and subsequently inhibiting E. coli growth. In our experiments, the addition of 1e4 mM each of four rNTPs gradually inhibited E. coli growth (Fig. 9B), indicating that the extra rNTPs indeed accelerated the lysis of T7-infected E. coli, possibly because of the promoted T7 DNA replication. In contrast, rNTPs (1e4 mM each) promoted E. coli growth in the absence of T7. Therefore, the addition of extra rNTPs inhibited E. coli growth, possibly due to the increased T7 propagation and the accelerated T7 DNA replication. 4. Discussion In most studies concerning DNA replication in vitro, the effects of rNTPs were ignored. In the studies that involve rNTPs or rNMPs show that rNTPs in solution and rNMP at primer terminus or embedded in template generally do not affect, inhibit, or even block DNA replication. The adverse effects can be summarized into three reasons. (i) rNTPs in solution chelates free Mg2þ ions and reduces its apparent concentration in solution for DNA polymerization. (ii) rNTPs could directly compete with dNTPs at polymerase active site and inhibit DNA replication. (iii) rNTP or rNMP shows extra repulsive effect with active site of DNA polymerase and retards DNA polymerization. In this work, unexpectedly, we found that rNTPs in an appropriate rNTPs/dNTPs concentration ratio range can promote DNA replication by KF exo or Pol T7 (Fig. S1). However, DNA

32

Z. Zou et al. / Biochimie 167 (2019) 25e33

Fig. 8. The binding affinity of Pol T7 to primer/template was enhanced in the presence of four rNTPs. Sensorgrams for binding of increasing concentrations of Pol T7 to the immobilized 30-mer/62a-mer primer/template in buffer A in the (A) absence or (B) presence of 0.2 mM each of four dNTPs, or (C) in the presence of 0.2 mM each of four dNTPs and 4 mM each of four rNTPs. The arrows indicate the beginning and stop of injection of Pol T7 into the chip. D-F, The binding signal and polymerase concentration were fit to estimate the dissociation constants. The solid lines represent the fit curves. Representative data from three independent experiments are displayed. The error bars represent the standard deviation of three independent experiments.

Fig. 9. Growth curves of E. coli DH5a or T7-infected E. coli DH5a in the presence of four rNTPs. A, The effect of rNTPs on the growth of E. coli. E. coli (OD600 of 0.9, 40 mL) and 160 mL LB medium containing 0, 1, 2, or 4 mM each of four rNTPs (final concentration) was incubated in a 96-well plate at 37  C. B, The same 50 mL E. coli and 60 mL bacteriophage T7 (6.5  106 pfu) in LB medium containing 0, 1, 2, or 4 mM each of four rNTPs (final concentration) was incubated in a 96-well plate at 37  C. OD600 measurements were taken every 5 min and E. coli growth curves were recorded. Representative data from three independent experiments are displayed.

polymerization by Gp90 exo, Dpo4, or hPol ɩ is gradually inhibited with increasing rNTP concentrations in a dose-dependent manner. The maximal promotion for Pol T7 was observed at rNTPs/dNTPs concentration ratio of 20 (Fig. 1). This promotion is independent of polymerase families, steric gates, base selectivities, or sugar discriminations (Table S2). This promotion is also irrelevant to wildtype or exonuclease-deficient polymerase, polymerase or Mg2þ concentrations, or primer/template sequences (Fig. 2). This promotion is not due to the optimized apparent Mg2þ concentration (Fig. 3) or the direct incorporation of extra rNTPs into DNA (Fig. 4). This promotion for Pol T7 polymerization was dependent on the concentrations and types of rNTPs (Fig. 5). Kinetic analysis shows that this promotion is originated from the increased fraction of productive complex and the accelerated DNA polymerization (Fig. 6). More Pol T7and DNA complex is

formed (Fig. 7) and their binding affinity is also enhanced (Fig. 8). These results explain how rNTPs promote DNA polymerization. Since no structure information is available, the actual promotion mechanism is unknown. Possibly, this promotion might be related with the allosteric effect [38e41]. rNTPs might bind at some unknown sites of Pol T7, inducing polymerase conformational change, which enhances the binding of polymerase to DNA and accelerates DNA polymerization. However, this binding should be very weak, since high concentrations of rNTPs are required for this promotion. Crystal structures of T7 DNA polymerase in complex with DNA in the presence of rNTPs may validate this hypothesis. Additionally, whether rNTPs could also accelerate DNA replication by other DNA polymerases remains to be explored. The addition of rNTPs accelerates E. coli growth compared with that without rNTPs (Fig. 9A). In contrast, the addition of rNTPs inhibits T7-infected E. coli growth, possibly by accelerating T7 DNA replication and T7 propagation (Fig. 9B). This work provides novel insight that rNTPs can promote T7 DNA replication and accelerate the lysis of T7-infected E. coli. In conclusion, it was the first time that we found rNTPs could promote Pol T7- DNA replication. This promotion is dependent on rNTP concentrations and types, but it is unrelated with the optimized Mg2þ apparent concentration or the direct incorporation of extra rNTPs into DNA. This promotion is originated from the increased fraction of productive polymerase-DNA complex and the increased polymerization rate. The promotion for T7 DNA replication also accelerates the lysis of T7-infected E. coli. This study discovers new functions of rNTPs in the promotion of DNA replication, which is completely different from the traditional concept that rNTPs always inhibit DNA replication. Conflict of interest The authors declare no conflicts of interest in this article. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Z. Zou et al. / Biochimie 167 (2019) 25e33

Acknowledgments This work was supported by China Key Research and Development Program [2017YFC1002002], the Fundamental Research Funds for the Central Universities, and the Youth 1000 Talent Plan. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2019.09.002. References [1] Y. Su, M. Egli, F.P. Guengerich, Mechanism of ribonucleotide incorporation by human DNA polymerase h, J. Biol. Chem. 291 (2016) 3747e3756. [2] M. Astatke, K. Ng, N.D.F. Grindley, C.M. Joyce, A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 3402e3407. [3] P.H. Patel, L.A. Loeb, Multiple amino acid substitutions allow DNA polymerases to synthesize RNA, J. Biol. Chem. 275 (2000) 40266e40272. [4] J.A. Brown, Z.C. Suo, Unlocking the sugar "steric gate" of DNA polymerases, Biochemistry 50 (2011) 1135e1142. [5] S.M. Cerritelli, R.J. Crouch, The balancing act of ribonucleotides in DNA, Trends Biochem. Sci. 41 (2016) 434e445. [6] N.Y. Yao, J.W. Schroeder, O. Yurieva, L.A. Simmons, M.E. O'Donnell, Cost of rNTP/dNTP pool imbalance at the replication fork, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 12942e12947. [7] J.F. Ruiz, R. Juarez, M. Garcia-Diaz, G. Terrados, A.J. Picher, S. Gonzalez-Barrera, A.R.F. de Henestrosa, L. Blanco, Lack of sugar discrimination by human Pol m requires a single glycine residue, Nucleic Acids Res. 31 (2003) 4441e4449. [8] N.A. Cavanaugh, W.A. Beard, V.K. Batra, L. Perera, L.G. Pedersen, S.H. Wilson, Molecular insights into DNA polymerase deterrents for ribonucleotide insertion, J. Biol. Chem. 286 (2011) 31650e31660. [9] A.R. Clausen, S. Zhang, P.M. Burgers, M.Y. Lee, T.A. Kunkel, Ribonucleotide incorporation, proofreading and bypass by human DNA polymerase d, DNA Repair 12 (2013) 121e127. [10] D.L. Watt, E. Johansson, P.M. Burgers, T.A. Kunkel, Replication of ribonucleotide containing DNA templates by yeast replicative polymerases, DNA Repair 10 (2011) 897e902. [11] Z. Zou, Z. Chen, Y. Cai, H. Yang, K. Du, B. Li, Y. Jiang, H. Zhang, Consecutive ribonucleoside monophosphates on template inhibit DNA replication by T7 DNA polymerase or by T7 polymerase and helicase complex, Biochimie 151 (2018) 128e138. [12] A.R. Clausen, M.S. Murray, A.R. Passer, L.C. Pedersen, T.A. Kunkel, Structure function analysis of ribonucleotide bypass by B family DNA replicases, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 16802e16807. [13] A.Y. Goeksenin, W. Zahurancik, K.G. LeCompte, D.J. Taggart, Z. Suo, Z.F. Pursell, Human DNA polymerase ε is able to efficiently extend from multiple consecutive ribonucleotides, J. Biol. Chem. 287 (2012) 42675e42684. [14] H.D. Zhang, U. Bren, I.D. Kozekov, C.J. Rizzo, D.F. Stec, F.P. Guengerich, Steric and electrostatic effects at the C2 Atom substituent influence replication and miscoding of the DNA deamination product deoxyxanthosine and analogs by DNA polymerases, J. Mol. Biol. 392 (2009) 251e269. [15] E.G. Frank, J.P. McDonald, K. Karata, D. Huston, R. Woodgate, A strategy for the expression of recombinant proteins traditionally hard to purify, Anal. Biochem. 429 (2012) 132e139. [16] B. Liu, S. Gu, N. Liang, M. Xiong, Q. Xue, S. Lu, F. Hu, H. Zhang, Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3 '-5 ' exonuclease activity, Virus Genes 52 (2016) 538e551. [17] S. Gu, J. Xiong, Y. Shi, J. You, Z. Zou, X. Liu, H. Zhang, Error-prone bypass of O6methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1, DNA Repair 57 (2017) 35e44. [18] B. Liu, Q. Xue, S. Gu, W. Wang, J. Chen, Y. Li, C. Wang, H. Zhang, Kinetic analysis of bypass of O6- methylguanine by the catalytic core of yeast DNA polymerase

33

h, Arch. Biochem. Biophys. 596 (2016) 99e107. [19] S. Gu, Q. Xue, Q. Liu, M. Xiong, W. Wang, H. Zhang, Error-free bypass of 7,8dihydro-8-oxo-2'-deoxyguanosine by DNA polymerase of Pseudomonas aeruginosa phage PaP1, Genes 8 (2017) 18. [20] H.D. Zhang, R.L. Eoff, I.D. Kozekov, C.J. Rizzo, M. Egli, F.P. Guengerich, Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translesion synthesis past bulky N2-Alkylguanine adducts, J. Biol. Chem. 284 (2009) 3563e3576. [21] H. Zhang, R.L. Eoff, I.D. Kozekov, C.J. Rizzo, M. Egli, F.P. Guengerich, Structurefunction relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes, J. Biol. Chem. 284 (2009) 17687e17699. [22] J. Yang, B. Li, X. Liu, H. Tang, X. Zhuang, M. Yang, Y. Xu, H. Zhang, C. Yang, General misincorporation frequency: re-evaluation of the fidelity of DNA polymerases, Biochem. Biophys. Res. Commun. 496 (2018) 1076e1081. [23] C.J. Potenski, H.L. Klein, How the misincorporation of ribonucleotides into genomic DNA can be both harmful and helpful to cells, Nucleic Acids Res. 42 (2014) 10226eU10798. [24] S.A.N. McElhinny, D. Kumar, A.B. Clark, D.L. Watt, B.E. Watts, E.B. Lundstrom, E. Johansson, A. Chabes, T.A. Kunkel, Genome instability due to ribonucleotide incorporation into DNA, Nat. Chem. Biol. 6 (2010) 774e781. [25] Q. Xue, M. Zhong, B. Liu, Y. Tang, Z. Wei, F.P. Guengerich, H. Zhang, Kinetic analysis of bypass of 7,8-dihydro-8-oxo-2ꞌ-deoxyguanosine by the catalytic core of yeast DNA polymerase h, Biochimie 121 (2016) 161e169. [26] Z. Zou, Z. Chen, Q. Xue, Y. Xu, J. Xiong, P. Yang, S. Le, H. Zhang, Protein interactions in T7 DNA replisome facilitate DNA damage bypass, Chembiochem 19 (2018) 1740e1749. [27] H.D. Zhang, S.J. Lee, B. Zhu, N.Q. Tran, S. Tabor, C.C. Richardson, Helicase-DNA polymerase interaction is critical to initiate leading-strand DNA synthesis, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 9372e9377. [28] H. Zhang, Y. Tang, S.-J. Lee, Z. Wei, J. Cao, C.C. Richardson, Binding affinities among DNA helicase-primase, DNA polymerase, and replication intermediates in the replisome of bacteriophage T7, J. Biol. Chem. 291 (2016) 1472e1480. [29] U. Qimron, B. Marintcheva, S. Tabor, C.C. Richardson, Genomewide screens for Escherichia coli genes affecting growth of T7 bacteriophage, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 19039e19044. [30] S.J. Lee, C.C. Richardson, The linker region between the helicase and primase domains of the gene 4 protein of bacteriophage T7 Role in helicase conformation and activity, J. Biol. Chem. 279 (2004) 23384e23393. [31] J.M.E. Forslund, A. Pfeiffer, G. Stojkovic, P.H. Wanrooij, S. Wanrooij, The presence of rNTPs decreases the speed of mitochondrial DNA replication, PLoS Genet. 14 (2018) e1007315. [32] S. Tabor, C.C. Richardson, Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 4076e4080. [33] S. Tabor, H.E. Huber, C.C. Richardson, Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7, J. Biol. Chem. 262 (1987) 16212e16223. [34] H.D. Zhang, F.P. Guengerich, Effect of N2-Guanyl modifications on early steps in catalysis of polymerization by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W, J. Mol. Biol. 395 (2010) 1007e1018. [35] I.H. Chaudry, Does ATP cross the cell plasma membrane, Yale J. Biol. Med. 55 (1982) 1e10. [36] J.Q. Ye, B. van den Berg, Crystal structure of the bacterial nucleoside transporter Tsx, EMBO J. 23 (2004) 3187e3195. [37] A.W. Feldman, E.C. Fischer, M.P. Ledbetter, J.-Y. Liao, J.C. Chaput, F.E. Romesberg, A tool for the import of natural and unnatural nucleoside triphosphates into bacteria, J. Am. Chem. Soc. 140 (2018) 1447e1454. [38] M.F. Colombo, D.C. Rau, V.A. Parsegian, Protein solvation in allosteric regulation: a water effect on hemoglobin, Science 256 (1992) 655e659. [39] I. Wong, T.M. Lohman, Allosteric effects of nucleotide cofactors on Escherichia coli rep helicase DNA binding, Science 256 (1992) 350e355. [40] R. Eliasson, E. Pontis, X. Sun, P. Reichard, Allosteric control of the substrate specificity of the anaerobic ribonucleotide reductase from Escherichia coli, J. Biol. Chem. 269 (1994) 26052e26057. [41] M. Yokoyama, H. Mori, H. Sato, Allosteric regulation of HIV-1 reverse transcriptase by ATP for nucleotide selection, PLoS One 5 (2010) e8867.