Research paperConsecutive ribonucleoside monophosphates on template inhibit DNA replication by T7 DNA polymerase or by T7 polymerase and helicase complex
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 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], [3], [4]].
DNA polymerases from bacteria, yeast, or humans can incorporate rNTPs into DNA [[5], [6], [7]], although polymerases can distinguish ribo- and deoxyribo-sugars via steric gate [[8], [9], [10]]. DNA Pol ε 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 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 approximately 2000 rNMPs per daughter chromosome [15]. Human Pol δ misincorporates ∼1 rNTP per 2000 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 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], 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 [6,15]. E. coli DNA replisome pauses 4–30 fold longer at a rolling circle DNA substrate including a 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], [26], [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), 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 perform the leading-strand DNA synthesis (strand-displacement DNA synthesis) at the fork DNA substrate. The primase domain of gp4 synthesizes the diribonucleotide ppAC 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 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 (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 synthesis using DNA fork substrate.
Section snippets
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 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), and E. coli thioredoxin
rNMPs altered duplex configuration
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 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 configuration [35,36].
rNMP gradually inhibited DNA synthesis by T7 DNA polymerase or by polymerase and helicase complex
Primer extension past
Discussion
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 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 conformation, which is
Conclusion
DNA synthesis is gradually inhibited with increasing the number of templating rNMP. rNMPs 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 consecutive templating rNMPs gradually inhibit primer extension by T7 DNA polymerase and strand-displacement
Conflicts of interest
The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.
Author contributions
Z. Zou, and H. Zhang conceived and designed the experiments. Z. Zou performed the experiments. Z. Chen, Y. Cai, H. Yang, K. Du, B. Li, Y. Jiang, and H. Zhang analyzed the data. Z. Zou, and H. Zhang wrote the paper.
Acknowledgements
We acknowledge financial support by China Key Research and Development Program (2017YFC1002002), the Fundamental Research Funds for the Central Universities, National Natural Science Foundation of China (31370793 and 81422041), and the Youth 1000 Talent Plan.
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