single-stranded ss m13 mp18 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    TaKaRa m13mp18 rf
    Chain-formation activity of HLTF with the multiply primed <t>M13mp18</t> 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.
    M13mp18 Rf, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m13mp18 rf/product/TaKaRa
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    m13mp18 rf - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    92
    TaKaRa m13mp18 single stranded
    Chain-formation activity of HLTF with the multiply primed <t>M13mp18</t> 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.
    M13mp18 Single Stranded, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m13mp18 single stranded/product/TaKaRa
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    m13mp18 single stranded - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    90
    Promega hpa i
    Chain-formation activity of HLTF with the multiply primed <t>M13mp18</t> 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.
    Hpa I, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hpa i/product/Promega
    Average 90 stars, based on 82 article reviews
    Price from $9.99 to $1999.99
    hpa i - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    80
    Boehringer Mannheim double stranded ds m13 mp18
    Chain-formation activity of HLTF with the multiply primed <t>M13mp18</t> 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.
    Double Stranded Ds M13 Mp18, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/double stranded ds m13 mp18/product/Boehringer Mannheim
    Average 80 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    double stranded ds m13 mp18 - by Bioz Stars, 2020-09
    80/100 stars
      Buy from Supplier

    Image Search Results


    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.

    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: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

    Techniques: Activity Assay, Titration, Recombinase Polymerase Amplification

    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.

    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: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

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

    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).

    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: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

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

    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.

    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: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

    Techniques: Activity Assay, Titration, Recombinase Polymerase Amplification

    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.

    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: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

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

    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).

    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: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

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