Journal: Nature Biotechnology

Article Title: Improved cytosine base editors generated from TadA variants

doi: 10.1038/s41587-022-01611-9

Figure Lengend Snippet: a , In vitro 24-hour end-point deamination assay to detect relative A-to-I (hAAG + APE1) and C-to-U (USERII) deamination by BE4, ABE8.20, CABE-Ts, and CBE-Ts programmed with the same guide and acting on the same dsDNA substrate. Endonuclease V, Endo V; human Alkyl Adenine DNA glycosylase, hAAG; Apurinic/apyrimidinic Endonuclease 1, APE1. Error bars represent standard deviation from the mean (plotted) of three independent replicates. Data were normalized to untreated sample. Endo V detects both A-to-I and C-to-U deamination. b , Left, Single-turnover rates of A-to-I or C-to-U deamination of the same dsDNA substrate by BE RNP. Right, Single-turnover rates of nicking by BE RNP in the same experiment as shown on left. Pseudo-first order k app rate constants obtained by fitting to single exponential are reported (mean ± s.d., n = 3 independent replicates).

Article Snippet: The precipitated reaction was resuspended in water and split into four equal parts: untreated, treated with Endonuclease V as described, treated with USER II as described and treated with human Alkyl Adenine Glycosylase (hAAG; NEB 0313S) followed by AP Endonuclease 1 (APE1; NEB M0282L) according to manufacturer’s instructions.

Techniques: In Vitro, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: Orphan base preference in plant and bacterial AP endonucleases. ( A ) Opposite base preference in a single sequence context. APE1 (0.02 nM), ARP (40 nM), ExoIII (0.06 nM) or EndoIV (0.3 nM) were incubated with DNA substrates (20 nM) containing a native AP site opposite G (orange), A (green), T (yellow) or C (blue) in the sequence context 5′-A X G-3′ ( X = AP site). ( B ) Opposite base preference in different sequence contexts. DNA substrates (20 nM) containing a native AP site opposite G (orange) or C (blue) in four different sequence contexts were incubated with APE1 (0.01 nM), ARP (40 nM), ExoIII (0.06 nM) or EndoIV (0.9 nM). The reaction products were stabilized with NaBH 4 , separated by denaturing PAGE and detected by fluorescence scanning for quantification. Data are the mean and standard error from two independent experiments. Asterisks indicate significant differences in a two-tailed Student’s t -test (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Sequencing, Incubation, Fluorescence, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: Divergent conservation of two DNA intercalating residues in APE1 orthologs. ( A ) Domain organization in human APE1, Arabidopsis ARP and E. coli ExoIII. ( B ) Structural superimposition of the double DNA insertion loops of APE1–DNA complex (PDB code: 5DFF) and ARP–DNA complex (PDB code: 8KA3). ( C ) Multiple sequence alignment of 25 representative APE1 orthologs showing the positions of the two DNA intercalating residues. ( D ) Amino acid frequencies in the two DNA insertion loops in APE1 orthologs from 2386 bacteria, 436 archaea, 242 fungi, 182 plants and 462 metazoans displayed as sequence logos generated by WebLogo 3.

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Sequencing, Bacteria, Generated

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: The opposite base preferences of APE1 and ARP are switched by exchanging the identity of the residue invading the minor groove. Different mutant versions of APE1 (0.2 nM) or ARP (40 nM) were incubated with DNA substrates (20 nM) containing a synthetic AP site (THF) opposite G (orange), A (green), T (yellow) or C (blue) in the sequence context 5′-A X G-3′ ( X = AP site). The reaction products were separated by denaturing PAGE, and detected by fluorescence scanning for quantification. Data are the mean and standard error from two independent experiments.

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Residue, Mutagenesis, Incubation, Sequencing, Fluorescence

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: Orphan base-dependent dissociation of ARP from its reaction product. ( A ) Schematic diagram of labelled DNA duplexes used as probes in the electrophoretic mobility shift assay (EMSA). The orphan base is indicated as S (= G or C). ( B ) EMSA. Increasing concentrations of APE1 or ARPΔ139 were incubated at 25°C for 25 min with Fl-labelled probes (10 nM) containing G or C as orphan base. After nondenaturing electrophoresis, free DNA (F) and protein–DNA complexes (bound DNA; B) were detected by fluorescence scanning. ( C ) Quantification of the fraction of bound DNA. The fraction of bound DNA with G (orange) or C (blue) as orphan base is plotted against protein concentration. Data are the mean and standard error from two independent experiments.

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Electrophoretic Mobility Shift Assay, Incubation, Electrophoresis, Fluorescence, Protein Concentration

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: Binding affinity of WT and mutant versions of APE1 and ARPΔ139 for dsDNA substrates and products with different orphan bases

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Binding Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: Exchanging the identity of the residue invading the major groove is sufficient to interconvert the activities of APE1 and ARP in ssDNA. ( A ) Schematic diagram of labelled DNA molecules used as dsDNA and ssDNA substrates. The AP site (THF) is indicated in red. ( B ) Different mutant versions of APE1 (0.2 nM) or ARP (40 nM) were incubated with dsDNA (pink) or ssDNA (purple) substrates (20 nM). The reaction products were separated by denaturing PAGE, detected by fluorescence scanning and quantified. Data are the mean and standard error from two independent experiments.

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Residue, Mutagenesis, Incubation, Fluorescence

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: Affinity for ssDNA is largely determined by the residue that intercalates into the major groove. ( A ) EMSA. Increasing concentrations of WT APE1 or ARPΔ139 were incubated at 25°C for 25 min with a Fl-labelled ssDNA probe (10 nM). After nondenaturing electrophoresis, free DNA (F) and protein–DNA complexes (bound DNA; B) were detected by fluorescence scanning. ( B ) The fraction of DNA bound to WT and mutant versions of APE1 or ARPΔ139 is plotted against protein concentration. Data are the mean and standard error from two independent experiments.

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Residue, Incubation, Electrophoresis, Fluorescence, Mutagenesis, Protein Concentration

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: Binding affinity of WT and mutant versions of APE1 and ARPΔ139 for ssDNA

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Binding Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases

doi: 10.1093/nar/gkae1297

Figure Lengend Snippet: The residue that invades the major groove is critical for mammalian APE1 function in antibody CSR. The relative percentage of IgA on the surface of CH12F3_APE1 Δ/Δ/Δ ( A ), or CH12F3_APE1 Δ/Δ/Δ APE2 Y/Δ ( B ) cells was measured after transient transfection with different mAPE1 constructs and CIT stimulation during 72 h. Data are the mean and standard error from five (panel A) or three (panel B) independent experiments. Asterisks indicate significant differences in a two-tailed Student’s t -test (* P < 0.05; ** P < 0.01; *** P < 0.001). Protein expression of the different mAPE1 variants was determined by western blot (upper panels). ( C ) Representative flow cytometry profile of the IgA switching population of CH12F3_ APE1 Δ/Δ/Δ cells after 72 h of cell growth under CIT− or CIT+ conditions.

Article Snippet: Human APE1 complementary DNA (cDNA) was synthetized by codon optimization for expression in bacteria (GenScript).

Techniques: Residue, Transfection, Construct, Two Tailed Test, Expressing, Western Blot, Flow Cytometry