Additional file 1 of Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3)
Creators: name: Han, Pengjun
name: Fan, Huahao
name: Tong, Yigang
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Figure S1. Structural domain prediction using InterPro for IME199 DNAP. IME199 DNAP has a DNA-dir_DNA_pol_B_mt/vir (IPR004868) structural domain, which belongs to the typical DNA polymerase B family. Figure S2. Alignment of the IME199 DNAP amino acid sequence with other family B DNA polymerases. The alignment was generated by MAFFT [1] and ESPript [2]. IME199 DNAP: Enterococcus phage IME199 DNA polymerase (GenBank accession no. ALO80851.1); phi29 DNAP: Bacillus phage phi29 DNA polymerase (GenBank accession no. ACE96023.1); 44AHDJ DNAP: Staphylococcus phage phi44AHJD DNA polymerase (GenBank accession no. AF513032.1); C1 DNAP: Streptococcus phage C1 DNA polymerase (GenBank accession no. AAP42306.1); WP-2 DNAP: Lactococcus phage WP-2 DNA polymerase (GenBank accession no. AHZ10879.1); CPS2 DNAP: Clostridium phage CPS2 DNA polymerase (GenBank accession no. AWG96526.1). Identical amino acid residues on a red background. The conserved motif sequences are underlined in black. Figure S3. Coomassie brilliant blue stained SDS-PAGE (10%) showing purified wild-type IME199 DNAP and its mutants (~95 kDa). The same protein size marker (Blue Plus® V, TransGen, China) was used in the experiment. All purified recombinant proteins have a 6-His label on the N-terminal. 199P represents IME199 DNAP in the figure. Figure S4. Study on the correction function of IME199 DNAP. IME199 DNAP was incubated with primed-templates mismatched 3′-terminus for 20 min at 30°C and then analyzed on a 20% denaturing PAGE gel. “−” means no IME199 DNAP, and “+” means with IME199 DNAP. The primed-template (25/50 nt) substrate was made by hybridizing two oligonucleotide strands (25 nt oligonucleotide sequence: 5′FAM- TCCTAACGAGATTAGTTTTGCTGTT -3′, 50 nt oligonucleotide sequence 5′- CCCATACAAATAAACCAAAAAACAATACAGCAAAACTAATCTCGTTAGGA -3′). The primed-template (27/50 nt) substrate was made by hybridizing two oligonucleotide strands (27 nt oligonucleotide sequence: 5′FAM- TCCTAACGAGATTAGTTTTGCTGTTAA -3′, 50 nt oligonucleotide sequence 5′- CCCATACAAATAAACCAAAAAACAATACAGCAAAACTAATCTCGTTAGGA -3′). The primed-template (29/50 nt) substrate was made by hybridizing two oligonucleotide strands (29 nt oligonucleotide sequence: 5′FAM- TCCTAACGAGATTAGTTTTGCTGTTAACA -3′, 50 nt oligonucleotide sequence 5′- CCCATACAAATAAACCAAAAAACAATACAGCAAAACTAATCTCGTTAGGA -3′). Figure S5. Amino acid sequence analysis between IME199 DNAP and phi29 DNAP. Alignment between IME199 DNAP and phi29 DNAP was made using CLC Sequence Viewer 6, and the regions containing homologous sequences are shown. Numbers indicate amino acid sites. Green color indicates amino acid residues of IME199 DNAP that are similar to those of phi29 DNAP. The conserved motif sequences are underlined in black. Red stars indicate amino acid residues associated with 3′-5′ exonuclease activity in phi29 DNAP. Pink diamond indicates amino acid residues associated with polymerase activity in phi29 DNAP. Figure S6. The yield of rolling circle amplification. 25 ng of single-stranded M13mp18 DNA was amplified by 100 nM IME199 DNAP (A) or 10 U phi29 DNAP (B) at 30 °C in the presence of primers of different lengths. The yield of DNA was calculated using the equation Yield = v × c, where v is the reaction volume and c is the concentration of detected DNA. Primer sequences are shown in Table S2. Figure S7. Comparison of phi29 DNAP using 28bp primers with different G+C content for rolling circle amplification. 25 ng of single-stranded M13mp18 DNA was amplified by 10 U phi29 DNAP at 30 °C in the presence of primers with different G+C content. The yield of DNA was calculated using the equation Yield = v × c, where v is the reaction volume and c is the concentration of detected DNA. Primer sequences are shown in Table S2. Figure S8. Sequencing results of the lacZα gene on the pUC19 plasmid in the white clones. On the left is the lacZ gene sequence, and the numbers represent the base sites. On the right is a summary of the sequencing results of the pUC19 plasmid in 26 white clones. Figure S9. Comparison of IME199 DNAP amplified different G+C sequences. Amplification was performed at 30 ℃ using random hexamer primers. Gene1 and gene2 are linear double-stranded DNA with a length of about 1600bp. Table S1. Analysis of phage genomes with terminal inverted repeat sequences. Table S2: Sequences of the oligonucleotides that have been used in this study.