Journal: Nature Communications

Article Title: Involvement of G-quadruplex regions in mammalian replication origin activity

doi: 10.1038/s41467-019-11104-0

Figure Lengend Snippet: Creation of an ectopic DNA replication origin. a Isothermal differential spectra (IDS; left panel) and circular dichroism spectra (CD; right panel) of potential OGRE/G4 sequences fond in the vicinity of replication origins. All tested sequences form G4 structures, as indicated by the strong negative peak around 295 nm and the two positive peaks around 240 and 273 nm (for IDS), and the strong positive peak around 260 nm (CD). The sequences are provided in Supplementary Table . b Ori1 that contains an OGRE/G4 element 240 bp upstream of the DNA replication initiation site (IS) was inserted by Cas9-stimulated homologous recombination into an origin-free region on chromosome 11 in NIH 3T3 mouse cells. The insertion of the 1907 bp fragment (marked in violet) occurred thanks to the two 500 bp homology arms (orange and green) present on the insertion template. The position of the primers (P) (sequences in Supplementary Table ) used for the analysis of origin activity is also shown. c Ori1 activity in parental (control in black) and recombinant NIH 3T3 cells (in grey). As expected, a two-fold increase in DNA replication activity was detected in recombinant cells compared with parental cells, whereas the external origin Ori2 exhibited the same replication activity in both cell lines. Note that SNS activity was also detected at the 5′ and 3′ junctions of the insertion site, but not in the corresponding control regions. The background control regions Bcgd1 and 2 are located in origin-free regions; results are the mean ± SD of 3 independent experiments; p values were obtained using the two-tailed Student’s t test; * p ≤ 0.05, p > 0.05. d Analysis of DNA replication, using the DpnI digestion method and colony counting (“Methods” section), in an EBV episomal plasmid transfected in HEK293 cells that express EBNA1. DNA replication activity was assayed using EBV episomal plasmids that carry or not (Delta) the OriP origin, or a 500 bp fragment of Ori2 containing an OGRE/G4 element in the sense or antisense orientation. Results are the mean ± SD of 3–7 independent experiments; p values were obtained using the two-tailed Student’s t test; * p ≤ 0.05; ns not significant, p > 0.05

Article Snippet: Deletion of an OGRE/G4 from an endogenous replication origin was obtained by transfection of the MLM3639 plasmid expressing the Cas9 nickase (hCas9_D10A) ( https://www.addgene.org/41816/ ), two different MLM3639 plasmids to express gRNAs specific to the targeted regions (gRNA Ori1 delG4 1 F, gRNA Ori1 delG4 1 R, gRNA Ori1 delG4 2 F, gRNA Ori1 delG4 2 R; for sequences see Supplementary Table ), and the pBABE-puro vector encoding the puromycin resistance gene.

Techniques: Circular Dichroism, Homologous Recombination, Activity Assay, Control, Recombinant, Two Tailed Test, Plasmid Preparation, Transfection

Journal: Protein Engineering, Design and Selection

Article Title: Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

doi: 10.1093/protein/gzs027

Figure Lengend Snippet: Ribbon diagram of HCA II with the active site and mutated surface residues indicated in ball-and-stick. (a) HCA II in blue with the mutated Leu residues in red, the zinc is shown as a magenta sphere with the hydrophilic residues involved with catalysis shown in yellow ball and stick. For panels (b)–(d) wild type and TS2 was superimposed to show the differences, wild type is in blue and TS2 is in yellow ball-and-stick; (b) Leu100His variant and inferred H-bonds (based on distance and angles) are shown as black dashed lines, (c) Leu224Ser, (d) Leu240Pro and waters and H-bonds. This figure was generated and rendered in PyMOL (DeLano, 2002).

Article Snippet: Crystallization and X-ray crystal structure determination Site-specific mutations of HCA II were made by GenScript.

Techniques: Variant Assay, Generated

Journal: Protein Engineering, Design and Selection

Article Title: Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

doi: 10.1093/protein/gzs027

Figure Lengend Snippet: Differential scanning calorimetry measurements of the major unfolding transition ( T M in °C) of each TS mutant compared with wild-type HCA II and the Y7F mutant

Article Snippet: Crystallization and X-ray crystal structure determination Site-specific mutations of HCA II were made by GenScript.

Techniques: Differential Scanning Calorimetry, Mutagenesis

Journal: Protein Engineering, Design and Selection

Article Title: Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

doi: 10.1093/protein/gzs027

Figure Lengend Snippet: Ball-and-stick diagrams of wild type and stabilized variants of HCA II active sites. The Zn is shown as a magenta sphere and all the hydrophilic active site residues (and their counterparts in the mutants) are shown as sticks, waters are shown as red spheres. Residues and waters are as labeled and inferred H-bonds are shown as black dashed lines. Maps are omitted for clarity. Figures were generated and rendered with PyMOL (DeLano, 2002).

Article Snippet: Crystallization and X-ray crystal structure determination Site-specific mutations of HCA II were made by GenScript.

Techniques: Labeling, Generated

Journal: Protein Engineering, Design and Selection

Article Title: Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

doi: 10.1093/protein/gzs027

Figure Lengend Snippet: Maximal, pH independent values of the kinetic constants for catalysis by variants of HCA II measured by 18 O exchange

Article Snippet: Crystallization and X-ray crystal structure determination Site-specific mutations of HCA II were made by GenScript.

Techniques:

Journal: Protein Engineering, Design and Selection

Article Title: Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

doi: 10.1093/protein/gzs027

Figure Lengend Snippet: The rate constant R1/[E] (s−1) as a function of temperature for the interconversion of CO2 and bicarbonate catalyzed by variants of HCA II. Wild type = black filled square; TS1 = red filled rhombus; TS2 = dark blue triangle; TS3 = brown square; TS4 = light blue filled square and TS5 = green filled triangle. Data have been separated on the ordinate to avoid superposition of points. Solutions contained 10 mM of all species of CO2 and 100 mM HEPES at pH 7.6.

Article Snippet: Crystallization and X-ray crystal structure determination Site-specific mutations of HCA II were made by GenScript.

Techniques: