Journal: STAR Protocols

Article Title: DNA methylation protocol for analyzing cell-free DNA in the spent culture medium of human preimplantation embryos

doi: 10.1016/j.xpro.2023.102247

Figure Lengend Snippet:

Article Snippet: Klenow DNA polymerase (3′ – 5 ′ exo-, 50 U/μL) , TIANGEN , NG202-02.

Techniques: Recombinant, Lambda DNA Preparation, Methylation, Multiplex Assay, Software, Stripping Membranes, Microinjection

Journal: STAR Protocols

Article Title: DNA methylation protocol for analyzing cell-free DNA in the spent culture medium of human preimplantation embryos

doi: 10.1016/j.xpro.2023.102247

Figure Lengend Snippet:

Article Snippet: Klenow DNA polymerase (3′ – 5 ′ exo-, 50 U/μL) , TIANGEN , NG202-02.

Techniques: Concentration Assay

Journal: Molecular cell

Article Title: Ca 2+ -stimulated AMPK-dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection

doi: 10.1016/j.molcel.2019.04.003

Figure Lengend Snippet: (a) Co-IP of Exo1(WT)-Flag or Exo1(S746A)-Flag with 14-3-3s in cells treated with HU (2 mM) or H2O for 3 h.

Article Snippet: Because we noticed that recombinant Exo1-His protein expressed in Sf9 cells was already phosphorylated at S746, we de-phosphorylated resin-bound Exo1-His by incubation with Lambda Protein Phosphatase (NEB, P0753) for 30 min at 30°C.

Techniques: Co-Immunoprecipitation Assay

Journal: Molecular cell

Article Title: Ca 2+ -stimulated AMPK-dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection

doi: 10.1016/j.molcel.2019.04.003

Figure Lengend Snippet: (a) Effects of siRNA-mediated knockdown of Exo1 on S4/8-phosphorylation of RPA in WT or AMPKα-KO HeLa cells after treatment with HU (2 mM) or H2O for 4 h.

Article Snippet: Because we noticed that recombinant Exo1-His protein expressed in Sf9 cells was already phosphorylated at S746, we de-phosphorylated resin-bound Exo1-His by incubation with Lambda Protein Phosphatase (NEB, P0753) for 30 min at 30°C.

Techniques:

Journal: Molecular cell

Article Title: Ca 2+ -stimulated AMPK-dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection

doi: 10.1016/j.molcel.2019.04.003

Figure Lengend Snippet: (a) Sequence alignment between S746 or the corresponding site in human, mouse and Xenopus Exo1 and the consensus phosphorylation sites of AMPK and Chk1. X, any amino acid.

Article Snippet: Because we noticed that recombinant Exo1-His protein expressed in Sf9 cells was already phosphorylated at S746, we de-phosphorylated resin-bound Exo1-His by incubation with Lambda Protein Phosphatase (NEB, P0753) for 30 min at 30°C.

Techniques: Sequencing

Journal: Applied and Environmental Microbiology

Article Title: Biosynthesis of a Complex Yersiniabactin-Like Natural Product via the mic Locus in Phytopathogen Ralstonia solanacearum ^▿ †

doi: 10.1128/AEM.05198-11

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: When required, antibiotics were added at the following concentrations: 100 μg of ampicillin/ml, 25 μg of chloramphenicol/ml, and 50 μg of kanamycin/ml. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Strain or plasmid Relevant properties a Source or reference Strains R. solanacearum GMI1000 Wild-type strain 33 RS6 RSc1806::pHiK002 in GMI1000 This study E. coli TOP10 F − mcrA Δ( mrr – hsdRMS – mcrBC ) φ80 lacZ ΔM15 Δ lacX74 recA1 araD139 Δ( ara leu ) 7697 galU galK rpsL (Str r ) endA1 nupG Invitrogen BW25113 K-12 derivative: Δ araBAD Δ rhaBAD 6 BW25113-2 BW21533 harboring pIJ790 and pHiK001 This study Plasmids pJET1.2 Cloning vector; Amp r Fermentas pIJ790 Lambda-Red ( gam , beta , exo ), araC , rep101 (Ts); Cm r 22 pAphA-3 Kanamycin resistance cassette; Km r 27 pHiK001 pJET1.2 carrying 1.7-kb RSc1806 fragment This study pHiK002 pHiK001 carrying RSc1806::Km r This study Open in a separate window a Abbreviations: Amp r , ampicillin resistance; Cm r , chloramphenicol resistance; Km r , kanamycin resistance.

Techniques: Plasmid Preparation, Cloning

Journal: Molecular cell

Article Title: RPA phosphorylation inhibits DNA resection

doi: 10.1016/j.molcel.2019.05.005

Figure Lengend Snippet: (A) Schematic of the single-molecule resection assay. (B) Kymographs of BLM (magenta) and EXO1 (green) or BLM and DNA2 (green) resecting DNA. (C) BLM/EXO1 and (D) BLM/DNA2 velocities and processivities with and without RPA (N>50 for all experiments). Violin plots: dots represent the median; black bars show the interquartile range (thick bars) and 95% confidence intervals (thin bars). (E) BLM and EXO1 (magenta) resecting DNA in the presence of 1 nM RPA-RFP (cyan) for 10 minutes before switching to pRPA-GFP (green) for 40 minutes. (F-G) Velocities (left) and processivities (right) of individual (F) BLM/EXO1 (N=90) and (G) BLM/DNA2 (N=42) complexes before and after switching from RPA to pRPA. Gray bars: molecules that stopped within our experimental uncertainty. Dashed line is shown as a reference with a slope of m=1. (H) Schematic and quantification of a qPCR-based cellular resection assay of cells transfected with RPA2(wt), RPA2(8D), or RPA2(8A). Error bars: SEM from three biological replicates. (I) Quantification of HR by I-Scel–induced DSBs in cells transfected with RPA2(wt), RPA2(8D), or RPA2(8A). Error bars: SEM from four biological replicates. (not significant; ns, p>0.05; * , p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

Article Snippet: FLAG-BLM was incubated with biotinylated EXO1, two units of DNase I (NEB), and 20 ng of bovine serum albumin (BSA, Fisher Scientific) in Buffer A (25 mM Tris-HCl [pH 8.0], 100 mM NaCl, and 10% glycerol) for 30 minutes on ice.

Techniques: Resection Assay, Transfection

Journal: Molecular cell

Article Title: RPA phosphorylation inhibits DNA resection

doi: 10.1016/j.molcel.2019.05.005

Figure Lengend Snippet: (A) Schematic of RPA indicating key phosphorylation sites on RPA32. Bottom: RPA32 phosphorylation induces physical interactions with RPA70N. (B) BLM (PDB:4CGZ) interacts with RPA70N (PDB:4IPC) on RPA (PDB:4GOP) via at least two N-terminal acidic patches (Fan and Pavletich, 2012; Feldkamp et al., 2013; Kang et al., 2018; Newman et al., 2015). (C) Kymographs of BLM/EXO1 (left) and BLM/DNA2 (right) resecting DNA in the presence of 1 nM RPA for 10 minutes before switching to RPAΔN for 40 minutes. (D) Ratios BLM/EXO1 and (E) BLM/DNA2 velocities (left) and processivities (right) before and after switching to the indicated RPA variants: RPAΔN (ΔN; N=57 for BLM/EXO1, N=50 for BLM/DNA2), yeast RPA (y; N=55 for BLM/EXO1, N=44 for BLM/DNA2), RPA plus RPA70 inhibitor (wt+inhib; N=30 for BLM/EXO1, N=51 for BLM/DNA2), phosphomimetic RPA (pmRPA; N=54 for BLM/EXO1, N=36 for BLM/DNA2), and phosphorylated RPA(8A) (p(8A); N=54 for BLM/EXO1, N=37 for BLM/DNA2). Both velocity and processivity are compared to the ratios of wt RPA (cyan bar) and pRPA (green bar) from Figure 1F,​,G.G. (not significant; ns, p>0.05; * , p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

Article Snippet: FLAG-BLM was incubated with biotinylated EXO1, two units of DNase I (NEB), and 20 ng of bovine serum albumin (BSA, Fisher Scientific) in Buffer A (25 mM Tris-HCl [pH 8.0], 100 mM NaCl, and 10% glycerol) for 30 minutes on ice.

Techniques: Inhibition

Journal: Molecular cell

Article Title: RPA phosphorylation inhibits DNA resection

doi: 10.1016/j.molcel.2019.05.005

Figure Lengend Snippet: (A) BLM is a processive helicase whereas the helicase-dead BLM(K695A) does not move on DNA. (B) ATP concentration-dependent BLM velocity (top) and processivity (bottom) fit to Michaelis-Menten kinetics (red). Error bars: SEM. Fit parameters and 95% CI are indicated. (C) BLM (magenta) with 1 nM RPA or (D) with pRPA. Bottom: particle-tracking of the kymograph above highlighting pausing (black) and strand-switching (red, green) segments of the trajectory. (E) RPA stimulates BLM helicase initiation. This requires BLM-RPA70N interactions. RPA variants: wt RPA (wt; N=78), pRPA (p; N=53), pmRPA (pm; N=60), RPAΔN (ΔN; N=43), and yRPA (y; N=40). Error bars: S.D. as determined by bootstrap analysis (Efron and Tibshirani, 1993). (F) BLM strand-switching is suppressed via BLM-RPA70N interactions. (G) Distribution of BLM velocities and processivities with RPA variants. (not significant; ns, p>0.05; * , p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). (H) Model of RPA and pRPA regulation of BLM helicase and BLM in concert with either EXO1 or DNA2 nuclease.

Article Snippet: FLAG-BLM was incubated with biotinylated EXO1, two units of DNase I (NEB), and 20 ng of bovine serum albumin (BSA, Fisher Scientific) in Buffer A (25 mM Tris-HCl [pH 8.0], 100 mM NaCl, and 10% glycerol) for 30 minutes on ice.

Techniques: Concentration Assay

Journal: Molecular cell

Article Title: RPA phosphorylation inhibits DNA resection

doi: 10.1016/j.molcel.2019.05.005

Figure Lengend Snippet: (A) Kymograph showing EXO1 (green) stalling at a nucleosome (magenta) (N=40). (B) BLM pushes nucleosomes in the presence of RPA (N=46). (C) BLM/EXO1 (N=68) and BLM/DNA2 (N=50) (left) are able to move nucleosomes in the presence of wt RPA. In contrast, pRPA (right) induces stalling of both BLM/EXO1 (N=40) and BLM/DNA2 (N=35) at a nucleosome. (D) Outcomes of collisions for the indicated resectosome components. (E) Relative velocities and processivities of nucleosomes that encounter the indicated resectosome components. Values are normalized to the same enzymes on naked DNA. (not significant; ns, p>0.05; * , p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). (F) A summary of how RPA phosphorylation negatively regulates DNA resection on chromatin.

Article Snippet: FLAG-BLM was incubated with biotinylated EXO1, two units of DNase I (NEB), and 20 ng of bovine serum albumin (BSA, Fisher Scientific) in Buffer A (25 mM Tris-HCl [pH 8.0], 100 mM NaCl, and 10% glycerol) for 30 minutes on ice.

Techniques:

Journal: Molecular cell

Article Title: RPA phosphorylation inhibits DNA resection

doi: 10.1016/j.molcel.2019.05.005

Figure Lengend Snippet: Key Resources Table

Article Snippet: FLAG-BLM was incubated with biotinylated EXO1, two units of DNase I (NEB), and 20 ng of bovine serum albumin (BSA, Fisher Scientific) in Buffer A (25 mM Tris-HCl [pH 8.0], 100 mM NaCl, and 10% glycerol) for 30 minutes on ice.

Techniques: Produced, Affinity Purification, Recombinant, Mutagenesis, Expressing, Lambda DNA Preparation, Plasmid Preparation, Software, Microscopy, Avidin-Biotin Assay, Flow Cytometry, Real-time Polymerase Chain Reaction, Imaging