template dna molecule  (Thermo Fisher)

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Mlh1–Pms1 rearranges DNA in an ATP-dependent mechanism and is inhibited by a pre-existing nick. ( A ) Schematic for UV-based structural probing assay used to detect Mlh1–Pms1-dependent DNA–DNA crosslinks. Plasmid substrates were incubated with Mlh1–Pms1 in the presence or absence of ATP or ATPγS. Reactions were then irradiated, deproteinated, and analyzed by agarose gel. Quantifications reflect the percentage of faster migrating species relative to the total amount of DNA in each lane (see the ‘Materials and methods’ section for additional details). ( B ) Control experiment showing the effect of UV treatment on relaxed DNA with and without Mlh1–Pms1 (100 nM) in reactions containing 20 mM NaCl and proteinase K. ( C, D ) Effect of nucleotide on formation of the faster migrating DNA species captured by UV-crosslinking on relaxed homoduplex or relaxed heteroduplex. Where present, Mlh1–Pms1 is included at 100 nM, Msh2–Msh6 is included at a final concentration of 50 nM, and ATP or ATPγS was included at a final concentration of 0.5 mM in a buffer containing 150 mM NaCl. All lanes in panel (D) were treated with proteinase K. ( E ) Control experiment showing the effect of UV treatment on supercoiled DNA with and without Mlh1–Pms1 (100 nM) in reactions containing 20 mM NaCl and proteinase K. ( F–H ) Effect of nucleotide and a pre-existing nick on formation of the faster migrating DNA species captured by UV-crosslinking on supercoiled and homoduplex and heteroduplex containing a pre-existing nick as shown in Fig. . Reactions were performed as in panels (C) and (D). All lanes in panel (H) were treated with proteinase K. For all panels showing quantifications, bar graphs represent means with error bars representing standard deviations between experiments. Statistical significance was determined using unpaired t -tests with Welch’s correction (Prism 10). Significance is denoted above bars (* P <.05; ** P <.005; *** P <.0005; **** P <.0001; ns, not significant).

Article Snippet: Mlh1-FLAG-Pms1 and any associated DNA molecules were immunoprecipitated using M2 anti-FLAG magnetic beads (Pierce).

Techniques: Plasmid Preparation, Incubation, Irradiation, Agarose Gel Electrophoresis, Control, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Mlh1–Pms1 is more sensitive to ATP on supercoiled DNA than relaxed topologies. ( A–F ) Electrophoretic mobility shift assays using either supercoiled or relaxed DNA substrates at 25 mM NaCl. In lanes 3–8 for each Mlh1–Pms1 was included at 25, 50, 100, 200, 300, or 400 nM. When present ATP or ATPγS was 0.5 mM. Binding reactions were incubated at room temperature for 20 min. Experiments were conducted in triplicate, and representative gels are presented for each condition, illustrating consistent observations across all replicates. ( G ) Modified topology-dependent binding assay reported by Litwin, et al. Creation of topoisomer pool and assay conditions are described in the ‘Materials and methods’ section. We were able to resolve seven distinct topoisomers and assigned them arbitrary numbers 0–6 from low to high supercoiling based on relative migration in an agarose gel. The input, supernatants from each separation step, and material where denaturation was incomplete were analyzed by agarose gel. Lane numbers corresponding to analysis in panels (B) and (C) are given. ( H–I ) Representative agarose gel analysis of reaction material. Where included, the concentration of ATP or ATPγS was 0.5 mM. Two hundred nanomolar Mlh1–Pms1 was added in all reactions. ( J ) The amount of each topoisomer was calculated in the bound and unbound fractions and compared to the input. See for quantification details. Because a portion of the bound population resisted proteinase K treatment and heat denaturation, the apparent amount of bound DNA expressed in the plots is input minus the sum of the unbound population for each topoisomer. ( K ) ATP hydrolysis assay measuring pmol of ATP hydrolyzed in a 45 min time period. Reactions contained 400 nM Mlh1–Pms1 and 3.8 nM 4.3 kb pBR322 plasmid either supercoiled or relaxed was included where indicated. The mean and standard deviation for three replicates is reported. ( L ) ATP hydrolysis assays conducted by pre-binding 400 nM Mlh1–Pms1 to 3.8 nM supercoiled or relaxed pBR322 for 15 min, followed by the addition of ATP. Reactions were incubated at 37°C for 45 min. The mean and standard deviation of three replicates is reported. For all panels (J)–(L), showing quantifications, bar graphs represent means with error bars representing standard deviations between experiments. Statistical significance was determined using unpaired t -tests with Welch’s correction (Prism 10). Significance is denoted above bars (* P <.05; ** P <.005; *** P <.0005; **** P <.0001; ns, not significant).

Article Snippet: Mlh1-FLAG-Pms1 and any associated DNA molecules were immunoprecipitated using M2 anti-FLAG magnetic beads (Pierce).

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Modification, Topology Dependent Binding Assay, Migration, Agarose Gel Electrophoresis, Concentration Assay, Hydrolysis Assay, Plasmid Preparation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Non-B-form regions disrupt Mlh1–Pms1 activities. ( A ) Dinucleotide repeats were inserted into a 2.7 kb pUC18 plasmid to assess impact on Mlh1–Pms1 activities. Oligonucleotides used to construct the inserts are given in . ( B–D ) Electrophoretic mobility shift assays on supercoiled DNA substrates containing dinucleotide repeat regions. Where indicated, Mlh1–Pms1 concentrations are 25, 50, 100, 200, 300, and 400 nM. Binding reactions were incubated at room temperature for 10 min. Experiments are performed in triplicate and representative gels are included. ( E–G ) DNA binding to linear substrates containing a dinucleotide repeat sequence linearized by BsaI-Hfv2, which positions the dinucleotide repeat sequence in the center of the plasmid. For panels (E) and (F) Mlh1–Pms1 was included at 50, 100, 200, 300, and 400 nM. For panel (G), Mlh1–Pms1 was included at 25, 50, 100, 200, 300, and 400 nM. Experiments are performed in triplicate and representative gels are included. ( H ) Endonuclease assays on substrates containing either no repeat sequence, or a (AT) 21 or (GC) 21 repeat sequence. Mlh1–Pms1 was titrated at 10, 25, 50, 100, 150, and 200 nM. The average proportion of supercoiled DNA converted to nicked circular product from three replicates. Error bars are the standard deviation between replicates. Data were fit to a sigmoidal function describing cooperative activity. Representative images are in .

Article Snippet: Mlh1-FLAG-Pms1 and any associated DNA molecules were immunoprecipitated using M2 anti-FLAG magnetic beads (Pierce).

Techniques: Plasmid Preparation, Construct, Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Sequencing, Standard Deviation, Activity Assay

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Mlh1–Pms1 enhances Topoisomerase I and Gyrase, with distinct roles for ATP. ( A ) Schematic for assay probing for E. coli Gyrase enhancement. Relaxed substrate was prepared by incubating supercoiled DNA with a topoisomerase, then inactivating the topoisomerase, see the ‘Materials and methods’ section for details. The relaxed circular DNA was then incubated with Mlh1–Pms1 or Mlh1–Pms1 ATPase mutants (titrated at 50, 100, or 200 nM) in the presence of 1 mM ATP, followed by the addition of E. coli Gyrase. Reaction products were analyzed by agarose gel. ( B–D ) The average proportion of supercoiled product relative to the total amount of DNA in each lane is reported for triplicate experiments. Reactions were performed at 24 mM KCl. Error bars represent the standard deviation between experiments. ( E ) Schematic for assay probing E. coli Topo I enhancement. Supercoiled DNA was incubated with Mlh1–Pms1 (titrated at 50, 100, or 200 nM where indicated) in the presence or absence of 0.5 mM ATP or ATPγS, followed by the addition of Topo I from E. coli . Reaction products were analyzed by agarose gel. ( F ) Summary data from agarose gel analysis of assay described in panel (E) using wild-type Mlh1–Pms1. ( G ) Summary of agarose gel analysis of assay described in panel (E) using mlh1N35A-Pms1 or Mlh1-pms1N34A ATPase mutants. For panels (F) and (G), the average proportion of relaxed circular DNA product relative to the total amount of DNA in each lane is reported for triplicate experiments. Reactions were performed at 50 mM potassium acetate. Error bars represent the standard deviation between experiments. For quantified data, bar graphs represent means with error bars representing standard deviations between experiments. Statistical significance was determined using unpaired t -tests with Welch’s correction (Prism 10). Significance is denoted above bars (* P <.05; ** P <.005; *** P <.0005; **** P <.0001; ns, not significant).

Article Snippet: Mlh1-FLAG-Pms1 and any associated DNA molecules were immunoprecipitated using M2 anti-FLAG magnetic beads (Pierce).

Techniques: Incubation, Agarose Gel Electrophoresis, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Mlh1–Pms1 can rearrange DNA into a structure recognizable by a structure-selective endonuclease. ( A ) Schematic for structure-selective T7 endonuclease I stimulation assay (see the ‘Materials and methods’ section for additional details). ( B ) Reactions with relaxed plasmid DNA (generated by topoisomerase) contained 100 nM Mlh1–Pms1, 0.5 mM ATP or ATPγS, and 0.6 units of T7 endonuclease I where indicated. The amount of DNA linearized was determined from three independent experiments. ( C ) Reactions with nicked plasmid DNA (generated with Nt.BspQI) contained 100 nM Mlh1–Pms1, 0.5 mM ATP or ATPγS, and 0.6 units of T7 endonuclease I where indicated. All reactions contained 50 mM NaCl. For each condition, the fraction of nicked DNA converted to linear product was quantified across three independent experiments. ( D ) Reactions with supercoiled plasmid DNA contained 50 nM Mlh1–Pms1, 0.5 mM ATP or ATPγS, and 0.2 units of T7 endonuclease I where indicated. The fraction of supercoiled DNA converted to nicked open circular or linear product was quantified across four independent experiments. For all panels, bar graphs show mean values with error bars representing standard deviations. Statistical significance was determined using unpaired t -tests with Welch’s correction (Prism 10). Significance is denoted above bars (* P <.05; ** P <.005; *** P <.0005; **** P <.0001; ns, not significant).

Article Snippet: Mlh1-FLAG-Pms1 and any associated DNA molecules were immunoprecipitated using M2 anti-FLAG magnetic beads (Pierce).

Techniques: Plasmid Preparation, Generated

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Mlh1–Pms1 and RFC compete on nicked DNA substrates. ( A ) UV treatment of nicked DNA substrates results in linearization. ( B, C, E, F ) A 2.7-kb nicked plasmid, either homoduplex or heteroduplex (as indicated), was incubated with wild-type Mlh1–Pms1 or the mlh1-N34A-pms1-N35A mutant (100 nM), RFC (100 nM), PCNA (500 nM), and, where applicable, Msh2–Msh6 (50 nM), in the presence or absence of 0.5 mM ATP. No MnSO 4 was included in any reaction. All reactions included 150 mM NaCl. Samples were then UV-irradiated, deproteinated, and analyzed by agarose gel electrophoresis as in Fig. . ( D, G ) Quantification of DNA degradation, measured as loss of signal in the UV-treated DNA band relative to negative controls. Data represent mean values ± standard deviation from three independent experiments. Statistical significance was determined by unpaired t -tests with Welch’s correction in Prism 10 and is denoted above bars (* P <.05; ** P <.005; *** P <.0005; **** P <.0001; ns, not significant).

Article Snippet: Mlh1-FLAG-Pms1 and any associated DNA molecules were immunoprecipitated using M2 anti-FLAG magnetic beads (Pierce).

Techniques: Plasmid Preparation, Incubation, Mutagenesis, Irradiation, Agarose Gel Electrophoresis, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Mlh1–Pms1 preferentially incises DNA with a pre-existing nick when bound before RFC/PCNA. ( A ) Schematic of the phased endonuclease assay used to test how the timing of nick formation influences Mlh1–Pms1 activity. ( B ) Assay in which plasmid DNA was pre-incubated with Mlh1–Pms1 (75 nM) for 10 min before adding RFC (100 nM), PCNA (500 nM), Nt.BspQI (10 units), and 2.5 mM MnSO 4 , to initiate endonuclease activity in a buffer containing 20 mM KCl. ATP (0.5 mM) was included where indicated to test nucleotide effects. % DNA nicked was measured as loss of single stranded bands relative to negative controls in lanes 3 and 8. All lanes are n = 3. ( C ) Parallel assays performed as in panels (A) and (B) but with 200 nM Mlh1–Pms1. In one control, Nt.BspQI was omitted (light yellow), resulting in Mlh1–Pms1 prebinding before RFC/PCNA and MnSO 4 were added. In the second control, both Nt.BspQI and the two-phase design were omitted by adding RFC/PCNA and Mlh1–Pms1 simultaneously (purple, striped bars). Quantification shows mean values from three independent experiments. Statistical significance was determined as in other assays (see for representative gels and Mlh1–Pms1 titration data).

Article Snippet: Mlh1-FLAG-Pms1 and any associated DNA molecules were immunoprecipitated using M2 anti-FLAG magnetic beads (Pierce).

Techniques: Activity Assay, Plasmid Preparation, Incubation, Control, Titration

Journal: Nucleic Acids Research

Article Title: Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

doi: 10.1093/nar/gkaf1252

Figure Lengend Snippet: Model for DNA rearrangement in mismatch repair. Mlh1–Pms1 is recruited to mismatched DNA through interactions with Msh2–Msh6 and can compact DNA. When Mlh1–Pms1 recognizes a pre-existing nick, it can locally distort the DNA but does not promote global compaction. Instead, nick-bound Mlh1–Pms1 may facilitate PCNA loading. Once loaded, PCNA-associated Mlh1–Pms1 can interact with Mlh1–Pms1 proteins bound near the mismatch. ATP binding then stimulates a global DNA rearrangement that mimics a supercoiled-like structure, potentially allowing PCNA-bound Mlh1–Pms1 to bridge sites distant from both the mismatch and the pre-existing nick. PCNA interactions also stimulate Mlh1–Pms1 endonuclease and ATPase activities, which may destabilize and dissociate Mlh1–Pms1 from the DNA, leaving the substrate accessible for mismatch removal and subsequent repair.

Article Snippet: Mlh1-FLAG-Pms1 and any associated DNA molecules were immunoprecipitated using M2 anti-FLAG magnetic beads (Pierce).

Techniques: Binding Assay