circular m13mp18 ss dna  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples

doi: 10.1093/nar/gkab1117

Figure Lengend Snippet: HPLC analysis of an extract from an 11.5-day-old C57BL/6 mouse embryo using the dNTP confirmation assay. ( A ) The two chromatograms show a mock-treated sample and a sample treated with DNA polymerization reagents (DNApol). M13 DNA, primers and DNA polymerase were present only in the DNApol sample, but the treatments were otherwise identical. The arrows in the bottom chromatogram show the specific removal of dNTPs. Peaks c and g are unknown components in the cellular extracts. ( B ) The same chromatograms as in A but shown at a lower magnification to visualize the rNTPs used as internal loading controls. The Fast Protocol was used for the HPLC analysis.

Article Snippet: For the DNA polymerase assay, we used M13 mp18 ss-DNA (250 μg/ml) from New England Biolabs, Ipswich, MA, USA (#N4040S), random decamers from Thermo Fischer Scientific (#AM5722G), and Klenow Fragment Exo – (5 U/μl) from Thermo Fischer Scientific (#EP0421).

Techniques:

Journal: Nucleic Acids Research

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

doi: 10.1093/nar/gky943

Figure Lengend Snippet: Suppression of the chain-formation activity of HLTF by interaction with RFC and PCNA. ( A ) Schematic of the experiments. Proteins were sequentially assembled on multiply primed M13mp18 ssDNA tethered to magnetic beads, and ubiquitin ligase assays were performed under standard assay conditions with the protein-bound DNA on the magnetic beads. (B, C) Western blot analysis of the assembled proteins, and ubiquitin chains generated by DNA-bound HLTF ( B ) and his HLTF ΔN ( C ) using anti-RFC1 (upper panels), anti-PCNA (second panels), anti-HLTF (third panels), and anti-ubiquitin antibodies (bottom panels). ‘–’ represents omitted proteins. ‘ΔN’ in RFC represents a mutant RFC consisting of ΔN555 RFC1. ‘FA’ represents the his HLTF FA mutant. ‘ΔN’ in HLTF represents his HLTF ΔN . Each signal intensity (SI) (%) under the HLTF blotting panels in (B) and (C) indicates the relative intensity of HLTF signals after normalization as shown, and that under the ubiquitin blotting panels in (B) and (C) indicates the relative intensity of signals in each plot larger than 60 kDa after normalization as shown. ND, not determined because signal levels were indistinguishable from the background. Relative specific activity (%) was calculated as [SI (%) of ubiquitin blot]/[SI (%) of HLTF blot] × 100. ( D ) Schematic representation of the structures of RFC1 and HLTF and their mutants. ( E ) Alignment of putative APIM sequences of HLTF homologues. The accession numbers of the sequences were NP_001305864 ( H. sapiens ), NP_033236 ( M. musculus ), NP_001179215 ( B. taurus ), XP_005510651 ( C. livia ), XP_018117635 ( X. laevis ), and XP_005163433 ( D. rerio ). ( F ) Effects of HLTF and his HLTF ΔN on singly primed ss M13mp18 DNA replication with pol δ. Reaction mixtures containing RPA, RFC, PCNA, RAD6-RAD18, MMS2-UBC13, and ubiquitin in the presence or absence of E1 and HLTF as indicated, but lacking pol δ, were preincubated at 30°C for 1 min, and DNA synthesis was started by addition of pol δ. Reactions were performed at 30°C for 10 min. The amounts of HLTF and his HLTF ΔN were increased in the order of 0.55, 1.1, and 2.2 pmol. The reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. ‘–’ indicates omitted proteins. ( G ) The radioactivity of [α- 32 P]dCMP incorporated into DNA was measured and normalized to the levels without HLTF.

Article Snippet: An amount corresponding to 150 pmol nucleotides as ss M13mp18 DNA or 300 pmol nucleotides as ds M13mp18 DNA was immobilized onto a 10 μl suspension of streptavidin magnetic beads M280 (Dynabeads) in DNA-binding buffer [20 mM HEPES–NaOH (pH 7.5), 50 mM NaCl, 0.2 mg/ml BSA, 1 mM DTT, and ubiquitin (174 pmol)] by incubation at room temperature for 30 min, followed by two washes with the same buffer of 25 μl each using a Dynal magnet.

Techniques: Activity Assay, Magnetic Beads, Ubiquitin Proteomics, Western Blot, Generated, Mutagenesis, DNA Synthesis, Agarose Gel Electrophoresis, Radioactivity

Journal: Nucleic Acids Research

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

doi: 10.1093/nar/gky943

Figure Lengend Snippet: Effects of various types of DNA. Chain-formation activities of wild-type HLTF ( A – E ) and his HLTF ΔN ( F, G ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Poly(dA)-oligo(dT) is a 2:1 mixture of poly(dA) and 18-mer oligo(dT) as nucleotides. (B, F) The indicated DNA was titrated as shown. (C, G) Poly(dA) was annealed to 18-mer oligo(dT) modified with biotin at the 5′- or 3′-end at a 2:1 ratio as nucleotides. (D) Poly(dA) was annealed to 18-mer oligo(dT) modified with phosphate at the 5′-OH or 3′-OH at a 2:1 ratio as nucleotides. ( E ) Poly(dA) was annealed to 18-mer oligo(dT) with one (-C1), two (-C2), or four (-C4) additional dCs at the 3′-end at a 2:1 ratio as nucleotides. The same data with poly(dA)-oligo(dT) were plotted in graphs for the wild type (A–E) and for his HLTF ΔN (F, G) as controls. Error bars from at least two experiments are shown with the symbols. ( H ) DNA-binding assay. HLTF (upper panel) or his HLTF ΔN (middle panel) was incubated with M13mp18 ssDNA (ss) or dsDNA (ds) tethered with magnetic beads, or magnetic beads only (–), at 4°C for 2 min, and the beads were separated from the supernatants. Each fraction was analyzed by western blotting with an anti-HLTF antibody, and band intensities were measured. The relative values of binding fractions normalized by the amount of the input were plotted in a graph (bottom panel).

Article Snippet: An amount corresponding to 150 pmol nucleotides as ss M13mp18 DNA or 300 pmol nucleotides as ds M13mp18 DNA was immobilized onto a 10 μl suspension of streptavidin magnetic beads M280 (Dynabeads) in DNA-binding buffer [20 mM HEPES–NaOH (pH 7.5), 50 mM NaCl, 0.2 mg/ml BSA, 1 mM DTT, and ubiquitin (174 pmol)] by incubation at room temperature for 30 min, followed by two washes with the same buffer of 25 μl each using a Dynal magnet.

Techniques: Ubiquitin Proteomics, Modification, DNA Binding Assay, Incubation, Magnetic Beads, Western Blot, Binding Assay

Journal: Nucleic Acids Research

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

doi: 10.1093/nar/gky943

Figure Lengend Snippet: Chain-formation activity of HLTF with the multiply primed M13mp18 ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.

Article Snippet: An amount corresponding to 150 pmol nucleotides as ss M13mp18 DNA or 300 pmol nucleotides as ds M13mp18 DNA was immobilized onto a 10 μl suspension of streptavidin magnetic beads M280 (Dynabeads) in DNA-binding buffer [20 mM HEPES–NaOH (pH 7.5), 50 mM NaCl, 0.2 mg/ml BSA, 1 mM DTT, and ubiquitin (174 pmol)] by incubation at room temperature for 30 min, followed by two washes with the same buffer of 25 μl each using a Dynal magnet.

Techniques: Activity Assay, Ubiquitin Proteomics, Titration

Journal: Nucleic Acids Research

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

doi: 10.1093/nar/gky943

Figure Lengend Snippet: Suppression of the chain-formation activity of HLTF by interaction with RFC and PCNA. ( A ) Schematic of the experiments. Proteins were sequentially assembled on multiply primed M13mp18 ssDNA tethered to magnetic beads, and ubiquitin ligase assays were performed under standard assay conditions with the protein-bound DNA on the magnetic beads. (B, C) Western blot analysis of the assembled proteins, and ubiquitin chains generated by DNA-bound HLTF ( B ) and his HLTF ΔN ( C ) using anti-RFC1 (upper panels), anti-PCNA (second panels), anti-HLTF (third panels), and anti-ubiquitin antibodies (bottom panels). ‘–’ represents omitted proteins. ‘ΔN’ in RFC represents a mutant RFC consisting of ΔN555 RFC1. ‘FA’ represents the his HLTF FA mutant. ‘ΔN’ in HLTF represents his HLTF ΔN . Each signal intensity (SI) (%) under the HLTF blotting panels in (B) and (C) indicates the relative intensity of HLTF signals after normalization as shown, and that under the ubiquitin blotting panels in (B) and (C) indicates the relative intensity of signals in each plot larger than 60 kDa after normalization as shown. ND, not determined because signal levels were indistinguishable from the background. Relative specific activity (%) was calculated as [SI (%) of ubiquitin blot]/[SI (%) of HLTF blot] × 100. ( D ) Schematic representation of the structures of RFC1 and HLTF and their mutants. ( E ) Alignment of putative APIM sequences of HLTF homologues. The accession numbers of the sequences were NP_001305864 ( H. sapiens ), NP_033236 ( M. musculus ), NP_001179215 ( B. taurus ), XP_005510651 ( C. livia ), XP_018117635 ( X. laevis ), and XP_005163433 ( D. rerio ). ( F ) Effects of HLTF and his HLTF ΔN on singly primed ss M13mp18 DNA replication with pol δ. Reaction mixtures containing RPA, RFC, PCNA, RAD6-RAD18, MMS2-UBC13, and ubiquitin in the presence or absence of E1 and HLTF as indicated, but lacking pol δ, were preincubated at 30°C for 1 min, and DNA synthesis was started by addition of pol δ. Reactions were performed at 30°C for 10 min. The amounts of HLTF and his HLTF ΔN were increased in the order of 0.55, 1.1, and 2.2 pmol. The reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. ‘–’ indicates omitted proteins. ( G ) The radioactivity of [α- 32 P]dCMP incorporated into DNA was measured and normalized to the levels without HLTF.

Article Snippet: To convert ss M13mp18 DNA into dsDNA, 650 ng of ss M13mp18 DNA annealed to the biotinylated primer was incubated with 10 U of T7 DNA polymerase (New England Biolabs) in a reaction buffer containing 0.33 mM each of dGTP, dATP, dTTP and dCTP at 37°C for 15 min.

Techniques: Activity Assay, Magnetic Beads, Western Blot, Generated, Mutagenesis, DNA Synthesis, Agarose Gel Electrophoresis, Radioactivity

Journal: Nucleic Acids Research

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

doi: 10.1093/nar/gky943

Figure Lengend Snippet: Effects of various types of DNA. Chain-formation activities of wild-type HLTF ( A – E ) and his HLTF ΔN ( F, G ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Poly(dA)-oligo(dT) is a 2:1 mixture of poly(dA) and 18-mer oligo(dT) as nucleotides. (B, F) The indicated DNA was titrated as shown. (C, G) Poly(dA) was annealed to 18-mer oligo(dT) modified with biotin at the 5′- or 3′-end at a 2:1 ratio as nucleotides. (D) Poly(dA) was annealed to 18-mer oligo(dT) modified with phosphate at the 5′-OH or 3′-OH at a 2:1 ratio as nucleotides. ( E ) Poly(dA) was annealed to 18-mer oligo(dT) with one (-C1), two (-C2), or four (-C4) additional dCs at the 3′-end at a 2:1 ratio as nucleotides. The same data with poly(dA)-oligo(dT) were plotted in graphs for the wild type (A–E) and for his HLTF ΔN (F, G) as controls. Error bars from at least two experiments are shown with the symbols. ( H ) DNA-binding assay. HLTF (upper panel) or his HLTF ΔN (middle panel) was incubated with M13mp18 ssDNA (ss) or dsDNA (ds) tethered with magnetic beads, or magnetic beads only (–), at 4°C for 2 min, and the beads were separated from the supernatants. Each fraction was analyzed by western blotting with an anti-HLTF antibody, and band intensities were measured. The relative values of binding fractions normalized by the amount of the input were plotted in a graph (bottom panel).

Article Snippet: To convert ss M13mp18 DNA into dsDNA, 650 ng of ss M13mp18 DNA annealed to the biotinylated primer was incubated with 10 U of T7 DNA polymerase (New England Biolabs) in a reaction buffer containing 0.33 mM each of dGTP, dATP, dTTP and dCTP at 37°C for 15 min.

Techniques: Modification, DNA Binding Assay, Incubation, Magnetic Beads, Western Blot, Binding Assay

Journal: Nucleic Acids Research

Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

doi: 10.1093/nar/gky943

Figure Lengend Snippet: Chain-formation activity of HLTF with the multiply primed M13mp18 ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.

Article Snippet: To convert ss M13mp18 DNA into dsDNA, 650 ng of ss M13mp18 DNA annealed to the biotinylated primer was incubated with 10 U of T7 DNA polymerase (New England Biolabs) in a reaction buffer containing 0.33 mM each of dGTP, dATP, dTTP and dCTP at 37°C for 15 min.

Techniques: Activity Assay, Titration