Article Cite This: Chem. Res. Toxicol. 2018, 31, 58−65
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Bypass of an Abasic Site via the A‑Rule by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1 Xiaoying Liu,†,‡ Xiaoli Zou,‡ Huangyuan Li,§ Zhenyu Zou,‡ Jie Yang,‡ Chenlu Wang,† Shunhua Wu,*,† and Huidong Zhang*,‡ †
School of Public Health, Xinjiang Medical University, Urumqi 830011, China Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan University, Chengdu 610041, China § Key Laboratory of Environment and Health among Universities and Colleges in Fujian, School of Public Health, Fujian Medical University, Minhou County, Fuzhou 350108, China ‡
S Supporting Information *
ABSTRACT: The abasic site is one the most common DNA lesions formed in cells; it induces a severe blockage of DNA replication and is highly mutagenic. We continue to use Gp90 exo−, the sole DNA polymerase from Pseudomonas aeruginosa phage PaP1, to study DNA replication upon encountering an abasic site lesion. Gp90 exo− can incorporate dNTPs opposite the abasic site, but extension past this site is extremely slow. Among the four dNTPs, dATP is preferentially incorporated opposite the abasic site, consistent with the A-rule. The incorporation is independent of the identity of the nucleotide 5′ of the abasic site. The incorporation of dATP opposite the abasic site occurs by direct incorporation of dNTP opposite the abasic site without a −1 frameshift deletion. Extension from an A:abasic site pair by Gp90 exo− is slightly unfavorable relative to those from other abasic site pairs. Incorporation of dATP opposite the abasic site is preferential and shows a biphasic shape, indicating that this incorporation is much faster than the subsequent dissociation of the polymerase from DNA. The template sequence does not affect the dATP incorporation priority, burst amplitude, burst rate, or dATP dissociation constant. Surface plasmon resonance shows that the presence of an abasic site in the template weakens the binding affinity of Gp90 exo− to DNA in a binary or ternary complex in the presence of any one kind of dNTP. This study reveals that Gp90 exo− preferentially inserts A opposite an abasic site via the Arule, like other DNA polymerases (e.g., Pol θ, KlenTaq, KF exo−, Pols α, δ/PCNA, and Thermococcus litoralis Pol Vent (exo−)), providing further insight into DNA replication mediated by P. aeruginosa phage PaP1 upon encountering an abasic site lesion.
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INTRODUCTION Accurate DNA replication is crucial for the maintenance of genomic integrity during cell proliferation and division.1 DNA replication is generally performed by high-fidelity DNA polymerases.2 However, this replication fidelity is under constant threat by a multitude of endogenous and exogenous factors that form various lesions within the genome and constitute an inevitable challenge for the replication machinery.3,4 Apurinic/apyrimidinic (AP) sites, referred to abasic sites, are produced by the spontaneous hydrolysis of an N-glycosyl bond or by hydrolysis of damaged bases by DNA glycosylases at a rate of ∼50 000 abasic sites/cell/day.5 Some environmental agents, such as methylmethanesulfonate, can also induce abasic site formation.6 The abasic site can lead to DNA replication being blocked as well as miscoding. An abasic site generally results in dATP insertion (A-rule),7 dGTP incorporation (Grule),8 and/or a −1 frameshift deletion.9 For example, human DNA Pol η is inefficient in abasic site bypass, and the majority of bypass events are A insertions.10−12 Human Pol β prefers to insert G opposite the abasic site.8 Sulfolobus solfataricus Dpo4 produces −1 frameshift deletions to a greater extent than A insertions.9 Yeast and human Pol η are very inefficient in both © 2017 American Chemical Society
inserting opposite of and extending from an abasic site, and they insert both A and G opposite the abasic site.6 LC−MS/MS sequence analysis of extension products of yeast Pol ηcore showed that 53% of the products contain dGTP misincorporation, 33% contain dATP, and 14% were −1 frameshifts, indicating that Pol ηcore bypasses an abasic site via a combination of the A-rule, the G-rule, and −1 frameshift deletion.13 Recently, our group identified that the sole DNA polymerase (Gp90) in Pseudomonas aeruginosa phage PaP1 is an A-family processive DNA polymerase containing 3′−5′ exonuclease activities on ssDNA and dsDNA.14 Exonuclease-deficient Gp90 exo− can bypass 8-oxoG in an error-free manner, but its incorporation efficiency is reduced.15 An alkylation lesion, O6MeG, partially inhibits full-length extension by Gp90 exo−, resulting in a 67-fold preferential misincorporation of dTTP rather than dCTP.16 Because abasic sites are one of the most common DNA lesions, we continue to use Gp90 exo− to further study the bypass of an abasic site, which is a continuation and extension of our recent work. In this work, Received: October 12, 2017 Published: November 28, 2017 58
DOI: 10.1021/acs.chemrestox.7b00287 Chem. Res. Toxicol. 2018, 31, 58−65
Article
Chemical Research in Toxicology
Figure 1. Extension assays performed by mixing 10 nM (A) wild-type Gp90 or (B) Gp90 exo−, 20 nM 32P-labeled 27-mer/62-mer dsDNA substrate containing G, AP1, or AP2, and 350 μM four dNTPs in buffer A as described in the Experimental Procedures. The numbers to the right depict the location of each substrate and product. Representative data from multiple experiments are shown. DNA substrate, and 350 μM each of all four dNTPs in buffer A (pH 7.5) containing 40 mM Tris-HCl (pH 7.5), 30 mM Mg2+, 10 mM DTT, and 50 mM potassium glutamate at 37 °C for 0.5, 1, 2, or 3 min. Reactions were terminated by addition of a quench solution containing 20 mM EDTA, bromphenol blue, 95% formamide (v/v), and xylene cyanol 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.15,16 Steady-State Kinetic Analysis of Single-Base Incorporation and Next-Base Extension. Steady-state kinetic analysis was performed by using Gp90 exo− and a 32P-labeled 27-mer or 28-mer primer annealed to a 62-mer template containing G or an abasic site (Table S1) in buffer A at 37 °C. The molar ratio of gp90 exo− to DNA substrate was
