crispr cas 9 homozygous erk1 ko  (Danaher Inc)

Journal: bioRxiv

Article Title: Novel DNA endonuclease activity of human ERK1 protein

doi: 10.1101/2025.03.03.641133

Figure Lengend Snippet: ( A ) ERK proteins (200 ng or 4.6 pmol per each reaction) with a constitutively active kinase function (R 84 S [ERK1m] and R 67 S [ERK2m], respectively) were compared for their activities to relax pBR322 in time course, 0, 10, and 30 min. Unless otherwise indicated, 250 ng (3.53 nM) of pBR322 was used per a reaction. SM, size marker; V line , pBR322 linearized by HindIII; CTRL, ERK1 storage buffer only control; OC/nick, open-circular or nicked plasmid DNA; Linear, linearized plasmid DNA; –SC, negatively supercoiled plasmid DNA. ( B ) Purified ERK1 (K1) and ERK1m (K1m) proteins were visualized by silver-staining and their identity confirmed by immunoblotting. ( C ) Relaxation assay-gel showing both K1 and K1m comparably relax supercoiled pBR322 in a dose-dependent manner (0, 18, and 180 nM). pBR322 relaxed by TOP2B (2.8 fmol) was included as a comparison. ( D ) Quantification of relaxation assay results with K1 (orange bars) and K1m (purple bars) as mean values (n = 3) and standard deviation (SD). P -values were calculated with the unpaired, one sided Student’s t-test. ns, non-significant. ( E ) Relaxation assay data with K1 and pBR322 in reactions ± ATP (0 or 1 mM final concentrations, green and yellow bars, respectively). The data were presented as mean values (n = 3) and SD. P -values were calculated with the unpaired, one sided Student’s t-test. ( F ) Relaxation assay data showing K1 dependence on Mg 2+ (Mg, 10 mM) for endonuclease activities. Zn 2+ and Ca 2+ were supplemented as 200 μM and 100 μM, respectively. Divalent ion final concentrations were determined considering their physiological concentrations. Note that linearized pBR322 DNA runs close to open circular one in ( C ) and ( F ) for the percentage of agarose gels (0.8%), compared to other gels (1.0 %).

Article Snippet: WT and CRISPR/Cas 9 homozygous ERK1 KO (1 bp deletion in exon 1) HEK293T cells were purchased from Abcam (Cat. ab266519).

Techniques: Marker, Control, Plasmid Preparation, Purification, Silver Staining, Western Blot, Comparison, Standard Deviation

Journal: bioRxiv

Article Title: Novel DNA endonuclease activity of human ERK1 protein

doi: 10.1101/2025.03.03.641133

Figure Lengend Snippet: ( A ) Relaxation assay-gel showing ERK1 (K1) concentration titration (0, 2.5, 25, 50, 125, and 250 nM) for 3 h. ( B ) Quantification of the relaxation assay data of K1 concentration titration (n ≥ 3). ( C ) Relaxation assay-gel showing pBR322 relaxation by K1 in time course analysis (0, 0.5, 1, 2, 3, 5 h). ( D ) Quantification of the relaxation assay data with K1 and pBR322 in time course analysis (n ≥ 3). ( E ) pBR322 map showing HindIII (Hind), NruI (Nru), and ori sites. ( F ) Agarose gels showing a single cut of pBR322 by either Hind or Nru. ( G ) K1-mediated linearized pBR322 (K1) was gel-extracted (GE), and digested by NruI (Nru) or HindIII (Hind). CTRL, pBR322. ( H ) A simple linear regression line and equation derived from the relation between DNA migration distance and molecular weights of size marker DNA fragments (Kbp). The visible bands, after each restriction enzyme cut the K1-mediated linearized pBR322, were calculated based on the equation.

Article Snippet: WT and CRISPR/Cas 9 homozygous ERK1 KO (1 bp deletion in exon 1) HEK293T cells were purchased from Abcam (Cat. ab266519).

Techniques: Concentration Assay, Titration, Derivative Assay, Migration, Marker

Journal: bioRxiv

Article Title: Novel DNA endonuclease activity of human ERK1 protein

doi: 10.1101/2025.03.03.641133

Figure Lengend Snippet: ( A ) Top, comparison of the N-terminal domain of ERK1 and ERK2. The region present only in ERK1 was boxed in sky-blue (11 – 27 a.a.). Bottom, R 15 (R15) and R 16 (R16) were marked with yellow. Three missense mutants with alanine substitutions were depicted as letter A (alanine) in red. ( B ) Purified R 15 A (15A), R 16 A (16A), and Double (R 15 A/R 16 A) mutant ERK1 proteins were visualized by silver-staining and confirmed by immunoblotting. ( C ) pBR322 relaxation by ERK1 mutants for 15 min in concentration titration (0, 60, 120, and 200 nM). ( D ) Quantification of reduced negatively supercoiled pBR322 bands by WT (black bars), 15A (blue bars), 16A (red bars), and Double mutant (purple bars) ERK1 in the concentration titration analyses (n = 3). ( E ) Three glutamates of E 13 , E 18 , and E 27 (marked with yellow) were substituted to alanines (A, marked in red) ( F ) Purified E 13 A (13A), E 18 A (18A), and E 27 A (27A) ERK1 proteins were visualized by silver-staining and confirmed by immunoblotting. ( G ) Concentration titration of WT, 13A, 18A, and 27A (0, 5, 10, and 20 nM; 15 min reaction time). ( H ) Quantification of increased OC or linear pBR322 bands by WT (grey), 13A (blue), 18A (yellow), and 27A (green) ERK1 in the concentration titration analyses (n = 3). ( I ) EMSA with pBR322 (1 nM) and WT, 13A, and 16A ERK1 (0, 1, 2.5, and 5 μM) in the absence of MgCl 2 , and ( J ) in the presence of MgCl 2 .

Article Snippet: WT and CRISPR/Cas 9 homozygous ERK1 KO (1 bp deletion in exon 1) HEK293T cells were purchased from Abcam (Cat. ab266519).

Techniques: Comparison, Purification, Mutagenesis, Silver Staining, Western Blot, Concentration Assay, Titration

Journal: bioRxiv

Article Title: Novel DNA endonuclease activity of human ERK1 protein

doi: 10.1101/2025.03.03.641133

Figure Lengend Snippet: ( A ) Left, AI-predicted ERK1 structure; middle, AI-predicted ERK2 structure; right, ERK1-ERK2 merged structures. ( B ) Left, 3D structure of ERK1 (green) and DNA (tan) complex in space-fills. Mg 2+ in dark blue. N 171 and D 184 in red sticks. Middle, the complex omitting space-fills and the area with a purple circle showing the putative catalytic site of ERK1 (silver) as an endonuclease and in a zoom-in view (right). Mg 2+ , a black sphere. Atomic distances (Å) shown in gray, dashed lines and numbers. ( C ) Merged crystal (green, PDB ID: 4H3Q) and AI-predicted (gold) ERK2 structures. ( D ) AI-predicted ERK2-DNA complex with a blue circle showing a Mg 2+ (A black sphere) binding site (left) and a zoom-in view (right). N 154 and D 167 shown in red stick. ( E ) Merged structures of WT (light gray), 13A (light blue), and 16A (light orange) ERK1. The E 13 A residue in 13A and the R 16 A residue in 16A were marked in pink and orange, respectively. NTD, N-terminal domain; Mg 2+ in black (WT), dark blue (13A), and red (16A); DNA binding cleft/domain boxed in red. ( F ) Comparison of distances between Mg 2+ and two coordinating amino acids, N 171 and D 184 in WT and 13A and 16A mutant ERK1 proteins. The same DNA (28-mer, 2292–2319 of pBR322) was used in Fig. 4, B, D , and F .

Article Snippet: WT and CRISPR/Cas 9 homozygous ERK1 KO (1 bp deletion in exon 1) HEK293T cells were purchased from Abcam (Cat. ab266519).

Techniques: Binding Assay, Residue, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Novel DNA endonuclease activity of human ERK1 protein

doi: 10.1101/2025.03.03.641133

Figure Lengend Snippet: ( A ) Diffraction-limited and reconstructed super-resolution multicolor images of ERK1 KO cells. Top, diffraction-limited (left) and super-resolution (right) fluorescence images. DAPI (red) and ERK1 (Alexa 647, green). Bottom, overexposed DAPI (DAPI°) merged with ERK1 (left) and the super-resolution image excluding oversaturated DAPI signals (right). F1–3, focal sites of DAPI and ERK1 overlapping signals. Intensity scales should be multiplied by 10 4 in arbitrary units (AU) throughout . ( B ) Zoom-in views of F1–3 showing cytoDNA-ERK1 interaction. ( C ) Validation of WT and ERK1 KO HEK293T cells by immunoblotting. β-ACTIN and HSP70 as reference and loading controls. ( D ) Cytoplasmic DAPI puncta (red) in an ERK1 KO HEK293T cell (left). V1–2 sites were magnified in diffraction-limited and super-resolution microscopy images (right). ( E ) Diffraction-limited and super-resolution microscopy images of WT and ERK1 KO cells showing colocalization of ERK1 (Alexa 647, green) and DNA (DAPI°, red). ( F ) Quantification of cytoplasmic puncta revealing a significant increase in ERK1 KO cells (blue violin, n = 38) compared to WT cells (pink violin, n = 17). The data were presented as mean values and SD. P -values were calculated with the unpaired, two-sided Student’s t-test.

Article Snippet: WT and CRISPR/Cas 9 homozygous ERK1 KO (1 bp deletion in exon 1) HEK293T cells were purchased from Abcam (Cat. ab266519).

Techniques: Fluorescence, Western Blot, Super-Resolution Microscopy