Journal: Genes and Environment

Article Title: Preparation of the standard cell lines for reference mutations in cancer gene-panels by genome editing in HEK 293 T/17 cells

doi: 10.1186/s41021-020-0147-2

Figure Lengend Snippet: Results of T7E1 assay. After transfection of expression vectors pRGEN_U6_SG, pRGEN-Cas9-CMV and ssODN corresponding to the target genes (KRAS, NRAS, PIK3CA, PTEN, BRAF and TP53), genomic DNA was isolated from the transfected cells. The target region was amplified by PCA with corresponding primers. After denature and re-annealing of the PCR product, it was digested by T7 endonuclease I (T7E1) which cut mismatched DNA fragments. Cleaved band suggests an introduction of mutations by ssODN

Article Snippet: Then 2 μl of T7 Endonuclease 1 (10 U/μl; New England Biolabs) was added and incubated at 37 °C for 30 min.

Techniques: Transfection, Expressing, Isolation, Amplification

Journal: Quarterly reviews of biophysics

Article Title: Nucleases: Diversity of Structure, Function and Mechanism

doi: 10.1017/S0033583510000181

Figure Lengend Snippet: The DEK superfamily consists of sequence- or structure-specific endonucleases and processive 3´ - 5´ exonucleases. a. The active site of BamHI contains (E)DEE, a variation of DEK. Except for panel b, the region containing the catalytic residues is shown in rainbow colored ribbon diagrams. The rest of the protein is colored grey. Divalent cations and the nucleophilic water are shown in purple and red spheres, respectively. b. Orthogonal views of BamHI-DNA interactions. The two BamHI subunits are shown in grey and light purple. The uneven β hairpins containing the DEK motif are highlighted in the rainbow colors. Only the β hairpins are shown when looking into the DNA major groove. BamHI cleaves DNA to form 4nt 5´ overhangs. The nucleotides containing the scissile phosphate are highlighted in pink sticks. c. Orthogonal views of HincII-DNA interactions. HincII cleaves DNA to blunt ends. The dimerization interface of HincII is nearly perpendicular to that of BamHI when looking down the DNA helical axis. d. E. coli MutH is a sequence- and methyl-specific endonucleaese with the DEK motif. The conserved Lys (K79) bridges the DNA sequence-specific binding in the major groove (shown in blue) and catalytic center approaching the minor groove (shown in pink). The divalent cations (Ca2+) are shown as green spheres. e. The trimeric phage λ exonuclease has a toroidal shape and degrades ssDNA processively. The three subunits are shown in grey, pink and blue ribbon diagrams. The active site region of the grey subunit is highlighted in rainbow colors. f. A zoom-in view of the active site with the eDEK motif. g. RecE, which is a processive exonuclease and promotes DNA recombination, is shown in the same color scheme as BamHI. h. The active site of RecB has the DEK motif with a His at its N-terminus like RecE. i. The Holliday-junction resolvase T7 endonuclease I has the (E)DEK motif like MutH and binds two metal ions. j. The structure specific endonuclease Mus81 and XPF also contain the DEK motif with two conserved carboxylates N-terminal to it. k. Vsr is a sequence-specific endonuclease. The DEK motif may be replaced by DHH, and the two metal ions in the active site are coordinated by a carbonyl oxygen (of T63) in addition to the conserved Asp and scissile phosphate. l. Dom3Z and Rai1 have the conserved DEK motif and can hydrolyze GTP.

Article Snippet: Relative to T7 endonuclease I, which has the REase-type active site and uses the two-metal-ion mechanism, T4 endo VII is not as specific for four-way junctions and can cleave 3-way junctions and mismatched DNA heteroduplexes, all of which are characterized by distorted double-helix structures ( Jensch & Kemper, 1986 ; Solaro et al., 1993 ).

Techniques: Sequencing, Binding Assay