Fujian Province Key Laboratory for the Development of Bioactive material from Marine Algae, Quanzhou Normal University, Quanzhou 362000, China
College of Life Sciences, Zhejiang University, Hangzhou 310058, China
The National Basic Research Program of China under contract No. 2015CB755903; the National Natural Science Foundation of China under contract Nos U1605214 and 31470133; the Foundation of Quanzhou Normal University under contract No. 2016YYKJ16.
DNA polymerase δ (Pol δ) plays a crucial and versatile role in DNA replication and DNA repair processes. Vent shrimp Rimicaris exoculata is the primary megafaunal community living in hydrothermal vents. In this study, the Pol δ from shrimp Rimicaris exoculata was cloned, expressed and characterized. The results showed that the Pol δ catalytic subunit (POLD1), 852 amino acids in length, shared high homology with crayfish Procambarus clarkii and shrimp Oratosquilla oratoria. The recombinant POLD1 expressed in Escherichia coli showed that the enzyme was active in a range of 20°C to 40°C with an optimum temperature at 25°C and in a wide range of pH with an optimum at pH 6.0. The activities of POLD1 were significantly enhanced in the presence of Triton-X 100, Tween 20 and Mn2+. The Km (dNTP) value of POLD1 was 4.7 μM. The present study would be helpful to reveal the characterization of Pol δ of deep-sea vent animals.
Figure 1. Characterization of vent shrimp POLD1. a. Nucleotide sequence and deduced amino acid sequence of shrimp R. exoculata POLD1. The functional domains of POLD1 were boxed. b. Phylogenetic analysis of POLD1 showing the positions of eight crustaceans used in the analysis. The tree was constructed by neighbor-joining method based on the protein sequence. The GenBank accession numbers were indicated in the parentheses. c. SDS-PAGE of expressed and purified proteins encoded by POLD1 gene of vent shrimp. Lanes M, protein molecular mass marker (kDa); 1. non-induced recombinant bacterium; 2. induced recombinant bacterium; 3. purified fusion protein His tag POLD1.
Figure 2. Effects of temperature on the activity and stability of POLD1. a. Optimum temperature of POLD1. DNA polymerase activity of vent shrimp POLD1 was assayed at various temperatures ranging from 15°C to 45°C. b. The thermostability of POLD1. The residual enzymatic activities were evaluated after incubation of the enzyme solution for 20 min, 40 min, 60 min or 80 min at 25°C, 30°C, 35°C, 40°C, 45°C or 50°C. In all panels, the maximum enzymatic activity was designated as 100%. Each point indicated the mean of triplicate assays within standard deviation.
Figure 3. Influence of pH on the DNA polymerase activity of POLD1. a. The range of pH values of vent shrimp POLD1. The DNA polymerase activity was examined at 25°C with different pH values ranging from 4.0 to 10.0. b. The DNA polymerase’s stability at different pH values. The recombinant polymerase was pre-incubated at a series of pH buffers (pH 5.0–10.0) for 20 min, 40 min, 60 min, or 80 min, followed by the DNA polymerase activity assay. The maximum enzymatic activity was designated as 100%. Each point indicated the mean of triplicate assays within standard deviation.
Figure 4. Effects of chelating agent, reducing agent, protease inhibitor, detergents and metal ions on the DNA polymerase activity of POLD1. a. Influence of inhibitors on the enzyme activity. The recombinant POLD1 was incubated with different inhibitors. Then the residual DNA polymerase activity was determined. The enzyme activity without inhibitors was defined as 100%. Each column indicated the mean of triplicate assays. b. Effects of metal ions on the DNA polymerase activity. The enzyme was incubated with different metal ions, followed by the examination of relative enzymatic activity. In all panels, the activity of enzyme without any treatment was designated as 100%. Each column represented the mean of triplicate within standard deviation. The significant differences between treatments and the control were indicated with asterisks (**, p<0.01).
Figure 5. Kinetic characterization of POLD1 using dNTP. The kinetic parameters Km and Vmax were estimated by linear regression from Lineweaver-Burk plots. All polymerase activity assays were conducted at 25°C and pH 6.5. Each point represented the mean of triplicate assays.