Consecutive ribonucleoside monophosphates on template inhibit DNA replication by T7 DNA polymerase or by T7 polymerase and helicase complex

Accepted Manuscript Consecutive Ribonucleoside Monophosphates on Template Inhibit DNA Replication by T7 DNA Polymerase or by T7 Polymerase and Helicase Complex Zhenyu Zou, Ze Chen, Ying Cai, Huang Yang, Ke Du, Bianbian Li, Yiguo Jiang, Huidong Zhang PII:

S0300-9084(18)30156-1

DOI:

10.1016/j.biochi.2018.05.022

Reference:

BIOCHI 5434

To appear in:

Biochimie

Received Date: 20 April 2018 Accepted Date: 31 May 2018

Please cite this article as: 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 (2018), doi: 10.1016/ j.biochi.2018.05.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Abtract rNTPs are structurally similar to dNTPs but are largely molar excessive in cell than dNTPs. rNTPs are inevitably incorporated into DNA to form rNMPs. Long RNA primers can

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also be incorporated into lagging-strand DNA. However, the influence of these incorporated rNMPs on T7 DNA replication remains unknown. In this work, we investigated primer

extension past consecutive rNMPs (rA, r(AC), r(ACC), or r(ACCA)) on template by T7 DNA

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polymerase or by its complex with helicase. Primer extension is gradually inhibited with

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increasing rNMP number. rNMPs decrease the dNTP incorporation efficiency, slightly weaken the binding affinity of polymerase to DNA in ternary complex, and reduce the protein interaction between polymerase and helicase at DNA fork, thereby decreasing the fraction of the productive enzyme·DNA complex and the average primer extension rate. Therefore, the

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consecutive rNMPs on template gradually inhibit T7 primer extension and strand-displacement DNA synthesis, providing the kinetic information for the inhibition of

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rNMPs on DNA replication.

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT Consecutive Ribonucleoside Monophosphates on Template Inhibit DNA Replication by T7 DNA Polymerase or by T7 Polymerase and Helicase Complex

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Zhenyu Zou1,2, Ze Chen1, Ying Cai2, Huang Yang2, Ke Du1, Bianbian Li1, Yiguo Jiang3, and Huidong Zhang1*

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From 1Public Health Laboratory Sciences and Toxicology, West China School of Public Health,

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Sichuan University, Chengdu, China. 2Institute of Toxicology, College of Preventive Medicine, The Third Medical University, Chongqing, PR China. 3Institute for Chemical Carcinogenesis, Guangzhou Medical University, Xinzao, Panyu District, Guangzhou, China.

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*To whom correspondence should be addressed at: Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan University, Chengdu, China. E-mail:

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[email protected]

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Running title: rNMPs inhibit T7 DNA replication

Keywords:

T7 DNA polymerase; DNA replisome; rNMP and rNTP; dNTP incorporation; ribonucleotide

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT Abtract rNTPs are structurally similar to dNTPs but are largely molar excessive in cell than dNTPs. rNTPs are inevitably incorporated into DNA to form rNMPs. Long RNA primers can also be

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incorporated into lagging-strand DNA. However, the influence of these incorporated rNMPs on T7 DNA replication remains unknown. In this work, we investigated primer extension past consecutive rNMPs (rA, r(AC), r(ACC), or r(ACCA)) on template by T7 DNA polymerase or by its complex with

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helicase. Primer extension is gradually inhibited with increasing rNMP number. rNMPs decrease the

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dNTP incorporation efficiency, slightly weaken the binding affinity of polymerase to DNA in ternary complex, and reduce the protein interaction between polymerase and helicase at DNA fork, thereby decreasing the fraction of the productive enzyme·DNA complex and the average primer extension rate. Therefore, the consecutive rNMPs on template gradually inhibit T7 primer extension and

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DNA replication.

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strand-displacement DNA synthesis, providing the kinetic information for the inhibition of rNMPs on

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT 1. Introduction Accurate copying of DNA by DNA polymerases is essential for cell propagation. High fidelity and proofreading activity of DNA polymerases ensure DNA replication at a low misincorporation

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frequency [1]. rNTPs and dNTPs have similar structures, except for a single OH group on the 2′ carbon of sugar in rNTPs. However, the concentrations of rNTPs are one to six orders of magnitude higher than the cellular dNTPs, depending on the cell type and stage of the cell cycle [2-4].

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DNA polymerases from bacteria, yeast, or humans can incorporate rNTPs into DNA [5-7],

although polymerases can distinguish ribo- and deoxyribo-sugars via steric gate [8-10]. DNA Pol ε

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incorporates more rNMPs while synthesizing the leading-strand DNA than Pol δ and Pol α synthesizing the lagging strand. Several DNA polymerases, such as terminal deoxynucleotidyl transferase and DNA polymerase µ, use rNTPs with a minimal preference for dNTPs [11, 12]. Pol β

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can incorporate rNTPs opposite normal bases or 8-oxoG [13]. rCMP is accumulated in the genome particularly opposite the modified guanines in the translesion DNA synthesis by Pol η [14]. Escherichia coli DNA polymerase III incorporates one rNMP for every 2.3 kb DNA and

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approximately 2,000 rNMPs per daughter chromosome [15]. Human Pol δ misincorporates ~ 1 rNTP

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per 2,000 dNTPs, resulting in >1 million rNMPs embedded in DNA after one replication cycle [5]. Although rNMPs can be removed through the proofreading function of DNA polymerases or by ribonuclease H2 enzymes [6], [16],[17], large numbers of ribonucleotides are present in the genomes of cells defective in the repair enzymes or RNase H2 [3, 18]. Except for these incorporated rNMPs by DNA polymerases, RNA primers with different lengths can be completely incorporated into the lagging-strand DNA to initiate the synthesis of Okazaki fragments [19]. Thus, if these incorporated rNMPs cannot be removed in good time, DNA polymerases may frequently encounter rNMPs on the

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT template strand. Any structure that differs from the standard nucleotides can be considered as DNA damage, including structural change in base or sugar ring. DNA damage in base, such as O6-MeG [20, 21],

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8-oxodG [22, 23], and abasic site [24], reduces the efficiency and fidelity of dNTP incorporation and even blocks DNA replication. The rNMP residues in DNA can alter the configuration of DNA duplex, pause DNA replication, make DNA susceptible to strand cleavages, and increase genome instability

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[6, 15]. E. coli DNA replisome pauses 4-30 fold longer at a rolling circle DNA substrate including a

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single rNMP relative to an unmodified substrate [15]. rNMPs in DNA template reduce the efficiency of DNA replication by S. cerevisiae DNA Pol α, δ, and ε [25-27].

In cells, DNA replisome synthesizes the leading- and lagging-strand DNA in a coordinated mode. Bacteriophage T7 has an efficient DNA replisome, which contains DNA polymerase (gp5/trx),

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helicase-primase (gp4), and ssDNA binding protein (gp2.5) [28, 29]. DNA polymerase synthesizes DNA with high processivity. The helicase domain of gp4 assembles as a hexamer and unwinds dsDNA to produce two ssDNA templates for DNA polymerases [30]. Polymerase and helicase

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perform the leading-strand DNA synthesis (strand-displacement DNA synthesis) at the fork DNA

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substrate. The primase domain of gp4 synthesizes the diribonucleotide pppAC and extends it to yield r(ACC), r(ACA), r(ACCC), r(ACAC), or (ACCA), and delivers them to DNA polymerase to initiate the Okazaki fragment synthesis [19]. The C-terminal tail of helicase interacts with the front basic patch (Fbp) and TBD basic patch (TBDbp) of DNA polymerase; while the non-tail region of helicase also interacts with DNA polymerase [28, 29]. These protein interactions maintain the fast and coordinated DNA replication [28, 29]. T7 DNA polymerase could incorporate rNTPs into DNA to form rNMPs on template. T7 primase 4

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT could also synthesize consecutive r(ACCA), r(ACAC), or r(ACCC) as primer to initiate the lagging-strand DNA synthesis. If these rNMPs cannot be removed efficiently, they will encounter T7 DNA polymerase. In this work, we will investigate the influence of one or several consecutive rNMPs

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(rA, r(AC), r(ACC), or r(ACCA)) on T7 DNA replication. These results show that rNMPs in DNA inhibit both the primer extension using primer/template substrate and strand-displacement DNA

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synthesis using DNA fork substrate.

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2. Materials and Methods 2.1 Materials

T4 polynucleotide kinase, dNTPs, and rNTPs were purchased from New England Biolabs (Beverly, MA). Non-hydrolyzable β,γ-CH2-dTTP was gift from Richardson′s lab at Harvard Medical

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School. [γ-32P] ATP (specific activity 3 × 103 Ci mmol-1) was from PerkinElmer Life Sciences (Boston, MA). Oligodeoxynucleotieds in Table S1 were synthesized and purified by HPLC (Takara Bio, Kyoto, Japan). Bacteriophage T7 exonuclease-deficient DNA polymerase (gp5 exo-), helicase-primase (gp4),

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and E. coli thioredoxin (trx) were overproduced and purified as described previously [29, 31]. Gp5

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exo- and 20-fold molar excess trx were pre-incubated at 4 oC for 10 min to form T7 DNA polymerase (gp5 exo-/trx). For simplicity, we denote gp5 exo-/trx as DNA polymerase and gp4 as helicase throughout the text. Other reagents were of the highest quality commercially available.

2.2 Primer Extension Past rNMPs on Template by T7 DNA Polymerase. The 23- or 27-mer ssDNA primer (P) was labeled with [γ-32P] ATP and then annealed with 62-mer template (T) containing dA, rA, r(AC), r(ACC), or r(ACCA) (Table S1) at molar ratio of 1:1.5 5

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT to form primer/template (P/T) DNA substrates. T7 DNA polymerase (5 nM) was incubated with 20 nM 27-mer/62-mer (standing start substrate, denoted as "S") or 23-mer/62-mer (running start substrate, denoted as "R") P/T DNA substrate for 5 min. Primer extension was initiated by mixing 300 µM of

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each of the four dNTPs with the pre-incubated enzyme/DNA mixtures in reaction buffer B (40 mM Tris-HCl (pH 7.5 at 37 oC), 10 mM MgCl2, 10 mM dithiothreitol and 50 mM potassium glutamate) at 37 oC. After reaction for 1 min, the mixture was terminated by addition of a quench solution

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containing 20 mM EDTA, bromphenol blue, 95% formamide (v/v) and xylene cyanol, and separated

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on a 20% polyacrylamide (w/v)/7 M urea gel. Products were visualized using a phosphorimaging screen and quantified by Quantity OneTM software [20-23]. Yields of the fully extended 62-mer product were calculated in percentage. The rNMP bypass capabilities were obtained by calculating the

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percentages of all products that were extended past all rNMPs.

2.3 Strand-Displacement DNA Synthesis Past rNMPs at Replication Fork by T7 DNA Polymerase and Helicase Complex.

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DNA replication fork (Fork) was prepared by annealing 5′-end 32P labeled 27-mer (or 23-mer)

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primer (P), 63-mer (or 67-mer) ssDNA flap (F) containing a 29 T tail at its 5′-side, and 62-mer template (T) containing dA, rA, r(AC), r(ACC), or r(ACCA) (Table S1) at molar ratio of 1:1.5:1.2. Helicase (120 nM, monomer concentration) was incubated with 20 nM DNA fork for 1 min; and then 60 nM T7 DNA polymerase was added for another 1 min to form helicase·polymerase·fork complex. The strand-displacement DNA synthesis was initiated by mixing 300 µM of each of the four dNTPs with the helicase·polymerase·fork complex at 37 oC. Then, the reaction was quenched and the products were separated, visualized, and quantified [20, 21] [22, 23]. The 62-mer product yields and 6

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT the bypass capabilities were calculated as described above.

2.4 Steady-State Kinetic Analysis of Single dNTP or UTP Incorporation Opposite dA or rA on

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Template. Single dNTP or UTP incorporation was performed by mixing 5 nM T7 DNA polymerase, 20 nM 32

P-labeled 27-mer/62-mer P/T DNA substrate, and varying concentrations of a single dNTP or UTP

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in the buffer B. The conversion of primer to product was kept < 20% [32]. Reactions were quenched

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and products were analyzed and quantified [20, 21]. The incorporation rates versus dNTP or UTP concentrations were fit to a hyperbolic Michaelis-Menten equation to yield kcat and Km values using GraphPad Prism (GraphPad, San Diego, CA). The standard errors were obtained from fitting using

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Prism. [32, 33].

2.5 Kinetic Analysis of Primer Extension Past rNMPs on P/T or at Fork Substrate. Rapid quench experiments were performed using a model RQF-3 KinTek quench flow apparatus

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(KinTek Corp, Austin, TX, USA) with 50 mM Tris-HCl buffer (pH 7.5) in both drive syringes and 0.5

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M EDTA solution in the middle quench syringe [21, 23]. Primer extension past rNMPs by DNA polymerase was performed by rapidly mixing 20 nM P/T substrate and 20 nM T7 DNA polymerase mixtures with an equal volume of 0.3 mM of each of the four dNTPs in buffer B at 37 oC. Strand-displacement DNA synthesis past rNMPs at fork by DNA polymerase and helicase complex was performed by rapidly mixing 20 nM fork DNA substrate, 60 nM T7 DNA polymerase, and 120 nM helicase (monomer concentration) mixtures with an equal volume of 0.3 mM each of the four dNTPs in buffer B at 37 oC. After incubation for the indicated time, the reactions were quenched by 7

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT 0.5 M EDTA. Substrate and product were separated; and the products were visualized and quantified [34]. The 62-mer product and time were fit to a single exponential Eq. 1: y = A (1 − ek t)

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(1)

where y is the 62-mer product concentration (nM), A is the amount of productive enzyme·DNA

from primer to 62-mer product (s−1), and t is time (s).

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complex that could extend primer to the full-length product (nM), k is the average rate of extension

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All nonlinear regression analysis and the standard errors were performed by GraphPad Prism [21]. Statistical significance was determined by GraphPad Prism. P < 0.05 was considered statistically significant.

Resonance (SPR).

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2.6 Binding Affinity of DNA Polymerase to Primer/Template Determined by Surface Plasmon

Binding of polymerase to 30-mer/62-mer P/T substrate was determined by SPR using a Biacore

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3000 instrument (Uppsala, Sweden) [29, 31]. The 30-mer primer contained a biotin at its 5′ terminus

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for coupling DNA onto a streptavidin (SA) chip and an Cdd (dideoxy-terminating cytidine) at its 3′-end for blockage of DNA polymerization. The template contained dA, rA, or r(ACCA) (Table S1). P/T (300 response units, RU) was immobilized onto the SA chip. Biotin was used in control cell to compensate for background. T7 DNA polymerase (80-800 nM) in buffer B in the absence or presence of 1 mM dTTP was flowed over the chip at a flow rate of 10 µl/min at room temperature. The binding signal and its corresponding T7 DNA polymerase concentration were fitted to Eq. 2 to estimate the dissociation constants Kd. The chip surface was regenerated by injection of 150 µl of 1 M NaCl at a 8

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT flow rate of 100 µl/min. Y = B × RUmax/(B + Kd)

(2)

where Y is the response signal corresponding to the binding (RU); B is concentration of T7 DNA

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polymerase (nM); RUmax is the maximal binding amount (RU); Kd is the dissociation constant (nM). All experiments were carried out thrice, and the standard errors were derived using GraphPad Prism.

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2.7 Binding Affinity of Polymerase or Helicase to Fork, and between Polymerase and Helicase at

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Fork.

DNA replication fork (30-mer/63-mer/62-mer containing dA, rA, or r(ACCA), 300 RU) were coupled to an SA chip. The primer contained a biotin at its 5′ terminus for coupling DNA onto the SA chip and an Cdd (dideoxy-terminating cytidine) at its 3′-end for blockage of DNA polymerization. T7

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DNA polymerase (0-1 µM) in buffer B containing 1 mM dTTP or helicase (0-3 µM, monomer concentration) in buffer B containing 1 mM non-hydrolyzable β, γ-methylene dTTP was flowed over the chip at a flow rate of 10 µl/min at room temperature. Non-hydrolyzable dTTP locked helicase

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around ssDNA strand at replication fork [30]. Biotin in control cell was used to compensate for

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background. The dissociation constants of polymerase or helicase from fork were determined using the same methods as described above [29, 31]. The same DNA fork (300 RU) were coupled to an SA chip. Helicase (1400 nM, monomer concentration) in buffer B containing 1 mM β, γ-methylene dTTP was flowed over the chip at 10 µl/min at room temperature to form a stably locked helicase·fork complex. Subsequently, T7 DNA polymerase (0-1000 nM) was injected in buffer B containing 1 mM β, γ-methylene dTTP. In control, the same DNA fork was immobilized without helicase to compensate for background. The binding 9

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT affinity of polymerase to helicase·fork complex was determined. Similarly, T7 DNA polymerase (500 nM) in buffer B containing 1 mM β, γ-methylene dTTP was flowed over the chip. Subsequently, helicase (0-5 µM, monomer concentration) was injected in buffer B containing 1 mM

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non-hydrolyzable dTTP. In control, the same DNA fork was immobilized without polymerase to compensate for background. The binding affinity of helicase to polymerase·fork complex was

Results

3.1 rNMPs Altered Duplex Configuration.

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3

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determined [29, 31].

The DNA configuration of 21-mer/62-mer duplex containing dA:dT, rA:dT, or r(ACCA):d(TGGT) base pairs was determined by circular dichroism spectroscopy (Fig. S1). The

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natural DNA duplex showed a conservative characteristic of B-form configuration. The signal intensity of DNA containing rA or r(ACCA) increased at 275 nm and decreased at 250 nm, thereby indicating that the B-form DNA duplex configuration was partially shifted to A-form RNA/DNA helix

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configuration [35, 36].

3.2 rNMP Gradually Inhibited DNA Synthesis by T7 DNA Polymerase or by Polymerase and Helicase Complex.

Primer extension past rNMPs by T7 DNA polymerase was performed using 27-mer/62-mer P/T "S" substrate or 23-mer/62-mer "R" substrate (Fig. 1A,B). DNA polymerase would encounter the first rA at the first incroproation position on the "S" substrate or encounter it at the 8th position after the initiation of primer extension using the "R" substrate. The efficiencies of primer extension on an 10

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT unmodified P/T substrate by DNA polymerase in the absence or presence of helicase were similar, showing the similar productive complex amounts (20 nM or 19 nM) and the average extension rates (1.6 min-1 or 2.6 min-1) (Fig. S2). The effects of rNMPs on primer extension were further investigated.

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Relative to the unmodified DNA, primer extension was gradually inhibited by rA, r(AC), r(ACC), or r(ACCA) using both "R" and "S" P/T substrates (Figs. 1A,B,E,F). The 28-mer and 29-mer extension products were produced corresponding to the incorporation of one and two nucleotides opposite the

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first r(A) or r(AC). Subsequently, the extension was inhibited by the rest of rNMPs on template.

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Strand-displacement DNA synthesis past rNMPs was performed by DNA polymerase and helicase complex using 27-mer/63-mer/62-mer "S" or 23-mer/67-mer/62-mer "R" DNA fork substrate (Fig.1 C,D). Polymerase was located at the 3′ end of the primer, and helicase encircled the 29 T tail of the DNA fork. Based on our previous work, helicase interacts with the front basic batch on DNA

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polymerase along its movement direction [29]; and T7 DNA polymerases are positioned in the behind of helicase at DNA replication fork [37]. DNA polymerase alone without helicase cannot perform the strand-displacement DNA synthesis using the fork DNA substrate, and only several nucleotides were

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extended [38, 39]. DNA polymerase and helicase complex could readily bypass dA, rA, or r(AC) with

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similar efficiencies but were inhibited by r(ACC) and r(ACCA) at the "S" fork (Fig. 1C,G). DNA polymerase and helicase complex could bypass all rNMPs at the "R" fork (Fig. 1D), but the extension was gradually inhibited with increasing rNMP number (Fig. 1H). The major 29-mer product corresponded to the incorporation of two nucleotides opposite r(AC) before blockage. Relative to the unmodified DNA, rNMPs partially or completely inhibited primer extension. The rNMP bypass capabilities (the percentages of all products that were extended past all rNMPs) were calculated and plotted against rNMP number in template for all the assays (Fig. 1I-L). The bypass 11

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT capability gradually decreased with increasing the rNMP number for both P/T and Fork DNA substrates.

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3.3 rNMPs Gradually Inhibited Full-Length Extension by Reducing the Productive Complex Amount and Average Extension Rate.

DNA synthesis past rNMPs involves multiple steps. To simplify this kinetic process, it can be

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summarized into two steps: (1) the binding step, DNA polymerase (+/- helicase) binds DNA substrate

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to form productive enzyme·DNA complex that could extend primer to products; and (2) the extension step, the productive complex extends the primer past rNMPs to the full-length 62-mer product. In some cases, only a fraction of DNA polymerase and DNA could form the productive complex to perform the DNA synthesis, due to the formation of non-productive complex as observed previously

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[32, 33]. The rNMPs on template may also affect the productive complex formation and its average extension rate. In this kinetic analysis, we used the molar excess polymerase and helicase relative to

product.

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DNA to expect that all DNA substrates were associated with these proteins and extended to 62-mer

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Primers could be fully extended past dA, rA, r(AC), r(ACC), or r(ACCA) by DNA polymerase using both "R" and "S" P/T substrates (Fig. 2A). The fully extended 62-mer product and time were fit to a single exponential equation (Fig. 2B) to estimate the fraction of productive polymerase·P/T complex (A) and the average extension rate (k) (Fig. 2C). The productive complex amounts gradually decreased with increasing rNMP number for both substrates. Both k values were similar for both substrates, but they gradually decreased with increasing rNMP number. The kinetic parameters of strand-displacement DNA synthesis were also determined using both 12

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT "R" and "S" fork DNA substrates. Similarly, molar excess polymerase and helicase relative to DNA fork were used. Primer could be fully extended past dA, rA, r(AC), r(ACC), or r(ACCA) for both DNA substrates (Fig. 3A). Certain intermediates (28-mer and 29-mer) were produced in the bypass of

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r(AC), r(ACC) and r(ACCA). The 62-mer product and time were fit to a single exponential equation to yield the fraction of productive polymerase·fork·helicase complex (A) and the average extension rate (k) (Figs. 3B,C,D). DNA polymerase alone without helicase cannot perform the

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strand-displacement DNA synthesis using the fork DNA substrate [38, 39]. Thus, only the

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pol-helicase-fork complex can be considered as the productive complex to fully extend the fork substrate. Both A and k values gradually decreased with increasing rNMP number for both fork substrates, indicating that rNMPs at fork gradually inhibit the strand-displacement DNA synthesis by

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T7 polymerase and helicase complex.

3.4 DNA Polymerase Tolerated a Templating rAMP but Significantly Discriminated against the Incoming rNTP.

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The steady-state kinetic parameters for dNTP or UTP incorporation opposite dA by DNA

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polymerase were measured (Table 1). dTTP was preferentially incorporated opposite dA and the misincorporation frequencies of the other three dNTPs were 10-5 - 10-6. In detail, kcat values were similar, but the Km of the three incorrect dNTPs significantly increased compared with that of dTTP. The efficiency of UTP incorporation opposite dA was reduced by 4,400-fold compared with that of dTTP, because of 104 higher Km value for UTP than for that of dTTP, indicating that T7 polymerase incorporated ribonucleotides very inefficiently. The dNTP or UTP incorporation opposite a templating rA by DNA polymerase were also studied 13

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT (Table 1). dTTP was preferentially incorporated opposite rA. The misincorporation frequencies of other three dNTPs were 10-4 - 10-5. The UTP incorporation efficiency opposite rA was reduced by 2,200-fold compared with that of dTTP. However, the efficiency of dTTP incorporation opposite rA

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decreased by only three fold compared with that of dTTP opposite dA. Therefore, DNA polymerase could tolerate a templating rA but significantly discriminated against the incoming rNTP.

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3.5 rNMPs Slightly Reduced the Binding Affinity of DNA Polymerase to P/T Substrate in Ternary

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Complex.

The dissociation constants (Kd,DNA) of DNA polymerase from 30-mer/62-mer P/T DNA substrate containing dA, rA, or r(ACCA) were determined by Surface Plasmon Resonance [21, 23]. In the absence of dTTP and Mg2+, DNA polymerase randomly bound to DNA to form a binary complex.

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Three Kd,DNA values were similar (300-340 nM, Fig. 4A), showing that one or four consecutive rNMPs on template did not affect the binding affinity of polymerase to DNA. DNA polymerase, DNA, and dTTP formed a ternary complex in the presence of dTTP and Mg2+. The Cdd (double deoxycytosine) at

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the 3′-end of primer blocked DNA polymerization. The Kd,DNA values showed a slight increase trend

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with increasing the rNMP number (Fig. 4B), indicating that rNMPs slightly weakened the binding affinity of polymerase to DNA in the ternary complex. Furthermore, the Kd,DNA values of the ternary complex were lower than those of the binary complex, showing that the presence of dTTP and Mg2+ significantly stabilized the binding of polymerase to DNA.

3.6 rNMPs Reduced the Binding Affinity between Polymerase and Helicase at the DNA Replication Fork. 14

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT The binding affinity of polymerase or helicase to DNA fork was also determined. DNA fork (30-mer/63-mer/62-mer) containing dA, rA, or r(ACCA) was immobilized on the SA chip. Helicase unwinds dsDNA using the energy from dTTP hydrolysis. The non-hydrolyzable β,γ-CH2-dTTP can

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lock helicase encircled ssDNA at replication fork. The Cdd (double deoxycytosine) at the 3′-end of primer blocked DNA polymerization. Varying concentrations of polymerase or helicase were flowed over the chip. The binding affinities of polymerase or helicase to the three DNA fork substrates

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showed a slight decrease trend with increasing the rNMP number (Kd,DNA of 400-720 nM or

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1100-1600 nM, Figs. 5A,B). Thus, one or four consecutive rNMPs at DNA fork slightly weakened the binding affinity of polymerase or helicase to DNA fork. Polymerase bound to the fork tighter than did helicase.

Subsequently, the binding affinity between polymerase and helicase at the DNA fork was also

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measured. DNA fork was coupled to the SA chip, and helicase was flowed over the chip to form a stable helicase·fork complex in the presence of β,γ-CH2-dTTP. Varying concentrations of polymerase was flowed over the chip to measure the binding affinities of DNA polymerase to helicase (Fig. 6A).

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Only the fork in the chip without helicase was used to compensate for background. The dissociation

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constants increased from 290 nM to 840 nM from dA to rA and r(ACCA), indicating that the rNMPs at DNA fork gradually reduced the binding affinity of polymerase to helicase. Similarly, the binding affinities of helicase to the polymerase·fork complex were also measured (Fig. 6B). The dissociation constants increased from 2600 nM to 5000 nM from dA to rA and r(ACCA), showing that rNMPs at fork gradually reduced the binding affinity of helicase to polymerase. Collectively, rNMPs at fork reduced the binding affinity between helicase and polymerase at the fork. Polymerase bound more tightly to the helicase·fork complex than did helicase to polymerase·fork complex, suggesting that the 15

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT DNA fork should first bind to helicase and then bind to polymerase to form a stable fork complex.

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Discussion

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T7 DNA polymerase could incorporate rNTPs into DNA to form rNMPs on template. T7 primase synthesizes consecutive r(ACCA) as primer to initiate the lagging-strand DNA synthesis. If these rNMPs cannot be removed in time, they will encounter T7 DNA polymerase or polymerase and

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helicase complex. A single rNMP residue can drive conformational equilibrium from B-form toward A-form [40]. The insertion of three consecutive rNMPs into dsDNA leads to the formation of A-form

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conformation, which is stabilized by the intramolecular interactions of the ribose 2′-hydroxyl groups [40]. We also found the presence of rNMPs in DNA shifts the B-form configuration to A-form. The change in DNA helix geometry may influence the efficiency and accuracy of DNA synthesis [41].

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Indeed, primer extension by T7 DNA polymerase was gradually inhibited by templating rA, r(AC), r(ACC), or r(ACCA) (Figs. 1A,B), similar to other results using S. cerevisiae DNA Pol α, δ, and ε [27], and yeast or human DNA Pol δ [5]. A templating rNMP reduces dNTP incorporation efficiency

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by three fold by T7 polymerase (Table 1) and nine fold by DNA Pol β [42]. Furthermore, E. coli DNA

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replisome pauses 4-30 fold longer at a rolling circle DNA substrate containing a single rNMP [15]. The progression is retarded by 2-8 fold by rNMPs using T7 DNA polymerase and helicase (Fig. 1H). A better understanding of the effects of rNMPs on DNA synthesis comes from the crystal structures of bacteriophage RB69 DNA polymerase bound to P/T containing rNMP. The 2′-oxygen of the ribose of rNMP displaces Tyr391 (palm subdomain) and disrupts its hydrogen bond with Tyr567 (fingers subdomain) [26], thus interfering the active site structure of DNA polymerase. The rNMP bypass capabilities (the percentages of all products that were extended past all rNMPs)

16

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT (Figs. 1I-L) gradually decreases with increasing the rNMP number for both P/T and fork DNA substrates. The amounts of productive enzyme·DNA complex (A values) and their average extension rates (k values) (Figs. 2 and 3) were normalized against the dA values obtained from the same kind of

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DNA substrate, and these values were plotted against the rNMP number (Fig. 7). The consecutive templating rNMPs gradually inhibits primer extension by polymerase alone and strand-displacement DNA synthesis by polymerase and helicase complex. Based on the kinetic analysis, the DNA

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synthesis can be summarized into two steps: the binding step (corresponding to A values) and the

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extension step (corresponding to k values). The A values decrease more significantly than do the k values with increasing the rNMP number. Thus, increasing rNMPs shows more adverse effect on the extension step than on the binding step.

The formation of the productive complex (A values) is related with the binding affinity of protein

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to DNA (Table 2). One or more templating rNMPs slightly weaken the binding affinity of DNA polymerase to P/T substrate in the ternary complex (Fig. 4B), reduce the binding of polymerase or helicase to fork substrate (Fig. 5), and obviously weaken the interactions between polymerase and

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helicase at DNA fork (Fig. 6). These weakened binding affinities may inhibit the formation of the

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productive enzyme·DNA complex and reduce the A values. The average extension rates (k values) are also gradually decreased with increasing rNMP number for both P/T and fork DNA substrates (Figs. 7C, D). T7 DNA polymerase and helicase move together at DNA fork in a coordinated mode. DNA synthesis by T7 polymerase provides the driving force to accelerate DNA unwinding by helicase and to push the strand-displacement DNA synthesis [38]. Thus, the average extension rates (k values) are dependent on the DNA synthesis. The efficiency of dTTP incorporation opposite rA decreases by three fold compared with that opposite dA, due to the 17

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT three-fold increase in Km value (Table 1), thus decreasing dTTP incorporation efficiency and reducing the average extension rates (k values). The binding of polymerase to P/T substrate (Kd of 50 nM, Fig. 4B) is much tighter than that of

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polymerase to helicase·fork complex (Kd of 300 nM, Fig. 6A). The binding data also shows that polymerase binds to the helicase·fork complex more tightly than does helicase to polymerase·fork complex (Fig. 6). Previously, we proposed that helicase should firstly bind to the ssDNA tail of DNA

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fork and then assist DNA polymerase to load onto the DNA fork [29], agreed with our results that the

polymerase·fork·helicase complex.

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fork substrate should first bind to helicase and then bind to polymerase to form a stable

Protein interactions in T7 DNA replisome facilitate DNA replication. In the initiation step, the Fbp of DNA polymerase interacts with the C-tail of gp4 to facilitate the loading DNA polymerase

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onto DNA fork [29]. In ongoing DNA synthesis, polymerase stably interacts with the non-C-tail region of helicase [29, 43]. When polymerase dissociates from the primer/template, the polymerase remains bound at the replication fork via the interaction of its TBDbp with the C-tail of helicase [29,

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44]. These protein interactions promote the strand-displacement DNA synthesis past rNMPs or other

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DNA lesion. T7 helicase could mediate DNA polymerase to bypass a nick [45]. DNA polymerase associated with helicase at DNA fork could directly replicate through a CPD lesion [46]. The protein interactions in the T7 DNA replisome could facilitate the bypass of 8-oxoG and O6-MeG at DNA fork [47]. The cycling of ssDNA binding (SSB) protein on and off DNA enables E. coli DNA replisome to bypass a large number of dimmer lesions produced by UV-irradiation [48]. All these results in T7 and other DNA replisome show that protein interactions at DNA replication fork could promote DNA synthesis past DNA damage. 18

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

5

Conclusion DNA synthesis is gradually inhibited with increasing the number of templating rNMP. rNMPs

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decrease dNTP incorporation efficiency, slightly reduce the binding affinities of polymerase to DNA in ternary complex, and weaken the binding between polymerase and helicase at DNA fork, thereby gradually decreasing the fraction of the productive complex and the average extension rate. Thus, the

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consecutive templating rNMPs gradually inhibit primer extension by T7 DNA polymerase and

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strand-displacement DNA synthesis by polymerase and helicase complex, providing the mechanism

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information for the inhibition effect of rNMPs on DNA replication.

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT Conflict of interest The authors declared no potential conflict of interest with respect to the research, authorship, and/or

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publication of this article.

Acknowledgements

We acknowledge financial support by China Key Research and Development Program

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(2017YFC1002002), the Fundamental Research Funds for the Central Universities, National Natural

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Science Foundation of China (31370793 and 81422041), and the Youth 1000 Talent Plan.

Author contributions

Z. Zou, and H. Zhang conceived and designed the experiments. Z. Zou performed the experiments. Z.

wrote the paper.

Abbreviation:

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Chen, Y. Cai, H. Yang, K. Du, B. Li, Y. Jiang, and H. Zhang analyzed the data. Z. Zou, and H. Zhang

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CD, circular dichroism spectroscopy; CPD, cyclobutyl pyrimidine dimmer; Fbp, front basic patch; gp4, gene 4 helicase-primase; gp5, gene 5 DNA polymerase exo-; rNMP, ribonucleoside

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monophosphate; rNTP, ribonucleoside triphosphate; dNTP, deoxyribonucleoside triphosphate; Pol, polymerase; P/T, primer/template; "R", running start substrate; RU, response unit(s); SA, streptavidin; "S", standing start substrate; SPR, surface plasmon resonance; TBDbp, the basic patches on the thioredoxin-binding domain; trx, thioredoxin.

20

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT for mismatch repair of leading-strand replication errors, Mol. Cell 50 (2013) 437-443. [19] H. Zhang, S.-J. Lee, C.C. Richardson, The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7, J. Biol. Chem. 287 (2012) 23644-23656.

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT [27] J.E. Stone, D. Kumar, S.K. Binz, A. Inase, S. Iwai, A. Chabes, P.M. Burgers, T.A. Kunkel, Lesion bypass by S. cerevisiae Pol ζ alone. DNA Repair 10 (2011) 826-834. [28] H.D. Zhang, S.J. Lee, C.C. Richardson, Essential protein interactions within the replisome

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT Table 1. Steady-state kinetic parameters for one-base incorporation opposite dA or rA by T7 DNA polymerase

rA

a

-3

-1

×10 min

Km,dNTP, µM

kcat/Km, -1

Misincorporation -1

frequencya

µM min

360 ± 30

(3.1 ± 0.1) ×10-3

120

dATP

300 ± 20

34 ± 1.3

8.9 × 10-3

7.4 × 10-5

dGTP

460 ± 40

57 ± 2.4

8.1 × 10-3

6.8 × 10-5

dCTP

400 ± 130

550 ± 17

7.2 × 10-4

6.0 × 10-6

UTP

820 ± 100

30 ± 1.9

2.7 × 10-2

2.8 × 10-4

dTTP

390 ± 40

(1 ± 0.1) × 10-2

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dATP

170 ± 10

7.8 ± 0.8

2.2 × 10-2

5.6 × 10-4

dGTP

400 ± 20

66 ± 1.2

6.1 × 10-3

1.6 × 10-4

dCTP

360 ± 10

400 ± 14

9.1 × 10-4

2.3 × 10-5

UTP

290 ± 10

1.8 × 10-2

4.7 ×10-4

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dTTP

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dA

kcat,

dNTP

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Template

16 ± 0.4

Misincorporation frequency is defined as (kcat/Km)other NTP/(kcat/Km)dTTP, where other NTP refers other dNTP or

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Table 2. The dissociation constants (Kd, nM) among helicase, polymerase, and DNA substrates (Fork + P/T + Pol Fork + Pol Fork + Helicase + (Fork + Pol) b Template P/T + Pol Helicase)a + + dTTP + dTTP β,γ-CH2-dTTP + Helicase Pol dA

300 ± 20

50 ± 4

400 ± 20

1100 ± 30

290 ± 23

2600 ± 130

rA

330 ± 24

67 ± 5

640 ± 25

1400 ± 65

510 ± 37

4200 ± 240

r(ACCA)

340 ± 33

80 ± 8

720 ± 40

1600 ± 96

840 ± 69

5000 ± 300

Binding of polymerase to helicase·fork complex.

b

Binding of helicase to polymerase·fork complex.

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a

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

Figure legends Fig. 1. DNA synthesis past dA, rA, r(AC), r(ACC), or r(ACCA) by T7 DNA polymerase or by

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polymerase and helicase complex. "S" and "R" DNA substrates and enzymes were shown at the top of the images. The numbers to the right depict the band location of each product.

P/T-Polassays were performed by mixing 5 nM T7 DNA polymerase, 20 nM 27-mer/62-mer ("S",

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A) or 23-mer/62-mer ("R", B) DNA substrate, and 300 µM each of the four dNTPs in buffer B for

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1 min. Fork-Pol-Helicase assays were performed by mixing 60 nM T7 DNA polymerase, 120 nM helicase (monomer concentration), 20 nM 27-mer/63-mer/62-mer ("S", C) or 23-mer/67-mer/62-mer ("R", D) fork substrate, and 300 µM each of the four dNTPs in buffer B for 1 min. E-H, The yields of the fully extended 62-mer product were quantified in percentage

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and plotted against rNMPs in template for each assay. I-L, The rNMP bypass capabilities (the percentages of all products that were extended past all rNMPs) were quantified and plotted for each assay. Representative data from three independent repetitions are illustrated. The error bars

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means standard error obtained from fitting using Prism.

Fig. 2. Kinetic analysis of primer extension past rNMPs on primer/template by DNA polymerase. A, Primer extension assays were performed with 20 nM T7 DNA polymerase, 20 nM 23-mer/62-mer ("R") or 27-mer/62-mer ("S") substrate, and 0.3 mM each of the four dNTPs in buffer B at 37 oC. B, The fully extended 62-mer product and time were fit to a single exponential equation. The solid lines represent the fit curves. C, Kinetic parameters (A, the productive complex amount; and k, the average extension rate) were listed. D, The productive complex 28

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT amounts and average extension rates were plotted against the rNMP number for both substrates. Representative data from three independent repetitions are illustrated. The error bars means

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standard error obtained from fitting using Prism.

Fig. 3. Kinetic analysis of strand-displacement DNA synthesis past rNMPs at DNA fork by DNA polymerase and helicase complex. A, The assays were performed with 60 nM T7 DNA

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polymerase, 120 nM helicase (monomer concentration), 20 nM 27-mer/63-mer/62-mer ("S") or

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23-mer/67-mer/62-mer ("R") fork substrate, and 0.3 mM each of the four dNTPs in buffer B at 37 oC. B, The fully extended 62-mer product and time were fit to a single exponential equation. The solid lines represent the fit curves. C, Kinetic parameters (A, the productive complex amount; and k, the average extension rate) were listed. D, The productive complex amounts and average

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extension rates were plotted against the rNMP number for both substrates. Representative data from three independent repetitions are illustrated. The error bars means standard error obtained

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from fitting using Prism.

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Fig. 4. Binding of DNA polymerase to primer/template containing dA, rA, or r(ACCA). Sensorgrams for binding of increasing concentrations of T7 DNA polymerase (0.08-0.8 µM) to the immobilized primer/template containing dA, rA, or r(ACCA) in buffer B in the (A) absence or (B) presence of dTTP and Mg2+. Polymerase binds to DNA to form a binary complex or form a ternary complex in presence of dTTP and Mg2+. The Cdd (dideoxy-terminating cytidine) at the 3′-end of primer could block DNA polymerization. The arrows indicate the beginning and stop of injection. The binding affinities of polymerase to DNA were determined using the steady-state 29

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT average response at each concentration of polymerase. The solid lines represent the theoretical curve calculated from the steady-state fit model (Biacore). Representative data from three

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independent experiments are presented.

Fig. 5. Binding of DNA polymerase or helicase to DNA fork containing dA, rA, or r(ACCA).

Sensorgrams for binding of increasing concentrations of (A) T7 DNA polymerase (gp5/trx, 0-1

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µM) with 1 mM dTTP or (B) helicase (0-3 µM, monomer concentration) with 1 mM

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non-hydrolyzable β, γ-methylene dTTP (for locking helicase on ssDNA) to the immobilized DNA fork containing dA, rA, or r(ACCA) in buffer B. The Cdd at the 3′-end of primer blocks DNA polymerization. The arrows indicate the beginning and stop of injection. The binding affinities of polymerase or helicase to DNA fork were determined. The solid lines represent the

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theoretical curve calculated from the steady-state fit model (Biacore). Representative data from three independent repetitions are displayed.

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Fig. 6. Binding of polymerase to helicase·fork complex or binding of helicase to polymerase·fork

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complex. A, Helicase (1.4 µM, monomer concentration) in buffer B containing 1 mM non-hydrolyzable β, γ-methylene dTTP was flowed over the chip containing the immobilized DNA fork to form stable helicase·fork complex. Polymerase (0-1000 nM) was injected in buffer B containing 1 mM non-hydrolyzable dTTP. The same DNA fork without helicase was used to compensate for background. The binding affinities of polymerase to the helicase·fork complex were determined. The solid lines represent the theoretical curve calculated from the steady-state fit model, and the arrows indicate the beginning and stop of injection. B, Similarly, DNA 30

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT polymerase (500 nM) in buffer B containing 1 mM non-hydrolyzable dTTP was flowed over the chip to form a stable polymerase·fork complex. The Cdd at the 3′-end of primer blocks DNA polymerization. Helicase (0-5 µM, monomer concentration) was injected in buffer B containing 1

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mM non-hydrolyzable dTTP to study the binding affinity of helicase to polymerase·fork complex. The same fork without polymerase was used to compensate for background. Representative data

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from three independent repetitions are displayed.

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Fig. 7. The productive complex amounts and average extension rates were normalized against the dA values obtained from the same kind of DNA substrate, and plotted against rNMP number for both P/T and fork DNA substrates. The error bars means standard error obtained from fitting

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using Prism. Asterisks indicate the statistically significant difference (P < 0.05).

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Fig. 1.

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

Fig. 2.

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

Fig. 3.

34

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

Fig. 4.

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Fig. 5.

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rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

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Fig. 6.

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RI PT

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

37

M AN U

SC

RI PT

rNMPs inhibit T7 DNA replication ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 7.

38

ACCEPTED MANUSCRIPT Highlights 1. The consecutive templating rNMPs gradually inhibit DNA synthesis.

2. rNMPs reduce the productive enzyme·DNA complex amount and average extension rate.

AC C

EP

TE D

M AN U

SC

4. rNMPs decrease the dNTP incorporation efficiency.

RI PT

3. rNMPs reduce the binding affinity between polymerase and helicase at DNA fork.