Journal: Nature Communications

Article Title: RNA-guided RNA silencing by an Asgard archaeal Argonaute

doi: 10.1038/s41467-024-49452-1

Figure Lengend Snippet: a HrAgo1 associates with 5’ phosphorylated (5’ P) small RNAs in vivo from E. coli . Nucleic acids that co-purified with HrAgo1 were [γ- 32 P] labeled, treated with RNase A or DNase I, and resolved on a denaturing gel (15% polyacrylamide 7 M urea). nt: nucleotides. b Length distribution of small RNAs associated with HrAgo1 as determined by small RNA sequencing. c Small RNAs associated with HrAgo1 have a bias for uracil bases at the 5’ end. d Sequences of guide and target oligonucleotides used in in vitro cleavage assays. e HrAgo1 cleaves ssRNA (but not ssDNA) targets with ssRNA guides, and ssDNA guides at lower efficiency, in the presence of Mg 2+ . HrAgo1 was incubated with ssDNA or ssRNA guides and Cy5-labeled ssDNA or RNA targets. Cy5-labeled cleavage products were resolved through denaturing (7 M urea) polyacrylamide gel electrophoresis and visualized by fluorescence imaging. Both ssRNA and ssDNA targets are 45nt. The HrAgo1-bound RNA extraction and digestion was carried out once; the results of the cleavage assays were confirmed by at least three repetitions.

Article Snippet: A plasmid suitable for expression of a HrAgo1 catalytic double-mutant (D585A & E623A; HrAgo1 DM ) was generated by Quikchange Site-Directed Mutagenesis using primers oPB199 and oPB201 for D585A and oPB200 and oPB198 for E623A, using E. coli strain NEB 5-alpha (New England Biolabs) (Table ). pX-sfGFP vector was a kind gift from Prof. Jae-Sung Woo (Korea University, South Korea).

Techniques: In Vivo, Purification, Labeling, RNA Sequencing Assay, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence, Imaging, RNA Extraction

Journal: Nucleic Acids Research

Article Title: DNA polymerase δ-dependent repair of DNA single strand breaks containing 3′-end proximal lesions

doi: 10.1093/nar/gkl1115

Figure Lengend Snippet: Repair ability of mouse embryonic fibroblasts deficient in Pol δ exonuclease against Hx located as a second nucleotide 5′- to a DNA single strand break. Plasmid constructs used in the experiments were prepared by ligation of the shown oligonucleotide duplexes into a luciferase reporter vector ( A ). Plasmids were transfected into either wild-type (Pol δ +/+) or mutant (Pol δ D400A) cells and after 8 h cell extracts were prepared and luciferase activity was measured. The average and standard error are shown graphically ( B ). Asterix (*) represents a statistical difference P < 0.02 compared to the wild-type cell line using t test.

Article Snippet: Antibodies against human Pol δ were raised in rabbit and affinity purified as described ( ) and antibodies against XPF were purchased from Abcam.

Techniques: Plasmid Preparation, Construct, Ligation, Luciferase, Transfection, Mutagenesis, Activity Assay

Journal: Nucleic Acids Research

Article Title: DNA polymerase δ-dependent repair of DNA single strand breaks containing 3′-end proximal lesions

doi: 10.1093/nar/gkl1115

Figure Lengend Snippet: Purification of the major activity against 5-OHU located as a second nucleotide 5′- to a DNA single strand break in human cells. A protein purification scheme was designed using HeLa cells ( A ) and involved generating whole cell extracts (step 1), separation of proteins by phosphocellulose chromatography using a step elution of 0.15 M KCl (PC-FI) and 1 M KCl (PC-FII; step 2), separation of proteins in PC-FII by Superose 12 gel-filtration chromatography (step 3), followed by separation of proteins (>100 kDa) using a Mono-Q column and a gradient elution of 0.05–1 M KCl (step 4). During each stage, the activity against a 5′-labelled 5-OHU lesion located as a second nucleotide 5′- to a DNA single strand break (5-OHU 2 ) was measured and the corresponding active fractions pooled, dialysed if necessary and used in the following step. Activity against 5-OHU 2 from fractions eluted from the final Mono-Q stage are shown ( B ) and these were further analysed by western blotting using XPF and Pol δ specific antibodies.

Article Snippet: Antibodies against human Pol δ were raised in rabbit and affinity purified as described ( ) and antibodies against XPF were purchased from Abcam.

Techniques: Purification, Activity Assay, Protein Purification, Chromatography, Filtration, Western Blot

Journal: Nucleic Acids Research

Article Title: DNA polymerase δ-dependent repair of DNA single strand breaks containing 3′-end proximal lesions

doi: 10.1093/nar/gkl1115

Figure Lengend Snippet: Immunodepletion of Pol δ from purified human whole cell extract fractions containing activity directed against 5-OHU 2 . Active fraction (fraction 22) from Mono-Q chromatography containing 5-OHU 2 activity was mock-immunodepleted and immunodepleted using Pol δ specific antibodies and samples analysed by SDS-polyacrylamide gel electrophoresis and western blotting using antibodies against XPF and Pol δ. ( A ) The original fraction, mock-immunodepleted and Pol δ immunodepleted fraction was tested for 5-OHU 2 activity using 300 fmol of duplex oligonucleotide ( B ) Samples were incubated for 20 min at 37°C prior to the addition of formamide loading dye and analysis by 20% denaturing polyacrylamide gel electrophoresis and phosphorimaging.

Article Snippet: Antibodies against human Pol δ were raised in rabbit and affinity purified as described ( ) and antibodies against XPF were purchased from Abcam.

Techniques: Immunodepletion, Purification, Activity Assay, Chromatography, Polyacrylamide Gel Electrophoresis, Western Blot, Incubation

Journal: Nucleic Acids Research

Article Title: DNA polymerase δ-dependent repair of DNA single strand breaks containing 3′-end proximal lesions

doi: 10.1093/nar/gkl1115

Figure Lengend Snippet: The 3′-5′-exonuclease activity of recombinant Pol δ against DNA lesions located as a second nucleotide 5′- to a DNA single strand break. Oligonucleotide substrates (0.3 pmol), shown at the top of each panel, were incubated with increasing concentrations of Pol δ (0–240 fmol) in reaction mixture without dNTPs for 20 min at 37°C prior to the addition of formamide loading dye. An aliquot was analysed by 20% denaturing polyacrylamide gel electrophoresis and phosphorimaging.

Article Snippet: Antibodies against human Pol δ were raised in rabbit and affinity purified as described ( ) and antibodies against XPF were purchased from Abcam.

Techniques: Activity Assay, Recombinant, Incubation, Polyacrylamide Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: DNA polymerase δ-dependent repair of DNA single strand breaks containing 3′-end proximal lesions

doi: 10.1093/nar/gkl1115

Figure Lengend Snippet: Reconstitution of repair of Hx located as a second nucleotide 5′- to a DNA single strand break using purified proteins. Hx 2 oligonucleotide substrate (0.3 pmol) was incubated with the indicated amount of either PCNA (3.5 pmol), Pol δ (240 fmol), FEN-1 (230 fmol) or DNA ligase I (10 fmol) in the presence of dNTPs for 20 min at 37°C prior to the addition of formamide loading dye. An aliquot was analysed by 20% denaturing polyacrylamide gel electrophoresis and phosphorimaging ( A ). The full complementation reaction (lane 8) was subsequently purified by phenol–chloroform extraction and Biospin P-30 gel filtration columns, incubated with MPG (600 fmol) and APE1 (600 fmol) for 20 min at 37°C prior to analysis by 20% denaturing polyacrylamide gel electrophoresis and phosphorimaging ( B ).

Article Snippet: Antibodies against human Pol δ were raised in rabbit and affinity purified as described ( ) and antibodies against XPF were purchased from Abcam.

Techniques: Purification, Incubation, Polyacrylamide Gel Electrophoresis, Extraction, Filtration

Journal: Nucleic Acids Research

Article Title: DNA polymerase δ-dependent repair of DNA single strand breaks containing 3′-end proximal lesions

doi: 10.1093/nar/gkl1115

Figure Lengend Snippet: Proposed mechanism of repair of DNA lesions located in close proximity to the 3′-end of a DNA single strand break by Pol δ. Complex DNA strand breaks containing 3′-proximal lesions that are resistant to the major BER enzymes are recognized by Pol δ (step A) that excises the lesion through its associated 3′-5′-exonuclease activity (step B). Pol δ is then able to insert the correct nucleotides into the gap causing strand displacement of the adjacent strand (step C). The subsequent 5′-flap generated is removed by FEN1 and DNA ligase I seals the nick (step D). PCNA can notably stimulate steps C and D.

Article Snippet: Antibodies against human Pol δ were raised in rabbit and affinity purified as described ( ) and antibodies against XPF were purchased from Abcam.

Techniques: Activity Assay, Generated

Journal: Journal of Clinical Microbiology

Article Title: Optimization of a Combined Human Parechovirus-Enterovirus Real-Time Reverse Transcription-PCR Assay and Evaluation of a New Parechovirus 3-Specific Assay for Cerebrospinal Fluid Specimen Testing

doi: 10.1128/JCM.01982-12

Figure Lengend Snippet: Comparison of CT values of EV, pan-HPeV, and HPeV3 assays with CSF clinical specimens tested in one-step and two-step RT-PCR reactions. (A) Two-step EV assay with the AGP kit enzyme versus one-step Cepheid enterovirus ASR (year 2008; n = 25); (B) two-step EV assay with the AGP kit enzyme versus one-step Argene enterovirus RUO assay (year 2009; n = 25); (C) two-step versus one-step EV assay with the AGP kit enzyme (year 2012; n = 30); (D) two-step versus one-step pan-HPeV assay with the AGP kit enzyme and historic two-step pan-HPeV assay with the ABI enzyme; (E) two-step versus one-step HPeV3 assay with the AGP kit enzyme and historic two-step HPeV3 assay with the ABI enzyme.

Article Snippet: The analytical specificity of the pan-HPeV assay reagents was tested against 13 enterovirus strains and other viruses obtained from ATCC: coxsackievirus types A9 (VR-186), B1 (VR-1032), B2 (VR-29), B3 (VR-30), B4 (VR-184), and B5 (VR-185); echovirus types 3 (VR-1040), 4 (VR-1041), 5 (VR-1043), 6 (VR-36), 7 (VR-37), 9 (VR-39), and 11 (VR-41); rhinovirus types 39 (ATCC VR-340), 8 (ATCC VR-488), 13 (ATCC VR-1123), 26 (ATCC VR-1136), and 27 (ATCC VR-1137); herpes simplex virus 1 (HSV-1) (ATCC VR-539) and HSV-2 (ATCC VR-540); human herpesvirus 6 (HHV-6; ATCC VR-1480); and laboratory-grown clinical isolates of cytomegalovirus (CMV) and Epstein-Barr virus (EBV).

Techniques: Comparison, Reverse Transcription Polymerase Chain Reaction