molecular weight size marker  (Bio-Rad)

Journal: bioRxiv

Article Title: Structural basis for the evolution of a domesticated group II intron-like reverse transcriptase to function in host cell DNA repair

doi: 10.1101/2025.01.14.632616

Figure Lengend Snippet: (A) Coomassie blue-stained NuPAGE 4-12% Bis-Tris gel of WT and RT3a mutant G2L4 RTs used in biochemical assay. Proteins were expressed with a N-terminal MBP tag and purified as described in Methods. The numbers to the left of the gel indicate molecular weights of a Color Prestained Broad Range (10-250 kDa) protein ladder (New England Biolabs). (B) Terminal transferase assay time courses with a 5’- 32 P-labeled 50-nt DNA substrate without a 3’-blocking group. Reactions were initiated by adding 1 mM of a single dNTP (dATP, dCTP, dGTP, and dTTP) and incubated at 37°C for times up to 30 min in reaction medium containing 10 mM Mg 2+ in the absence (top panels) or presence (bottom panels) of 1 mM Mn 2+ . The numbers to the left of the gel indicate the positions of 5’-labeled RiboRuler Low Range RNA Ladder size markers run in a parallel lane. The plots to the right of the gel show the average value and variance for two repeats of the experiment. Tables to the right of the plots show the rate constants (k obs ) and amplitudes (Ampl.) of labeled products >50 nt obtained by fitting to a first-order rate equation. Ampl. values in parentheses represent fixed amplitudes for reactions that did not reach an end point based on the average Ampl. value for those that reached a clear end point during the experiment. The red asterisk in the schematic at the bottom indicates a 5’- 32 P-label.

Article Snippet: Products were analyzed by electrophoresis on a non-denaturing 12% polyacrylamide gel against double-stranded DNA size markers (5’-labeled Low Molecular Weight DNA Ladder; New England Biolabs) with TBE (89 mM Tris, 89 mM Borate and 2 mM EDTA) buffer.

Techniques: Staining, Mutagenesis, Purification, Glutathione S-Transferase Assay, Labeling, Blocking Assay, Incubation

Journal: bioRxiv

Article Title: Structural basis for the evolution of a domesticated group II intron-like reverse transcriptase to function in host cell DNA repair

doi: 10.1101/2025.01.14.632616

Figure Lengend Snippet: (A) Size-exclusion chromatography of purified WT and RT3a mutant MBP-tagged G2L4 RTs. Elution profiles of different proteins are color-coded as indicated in the Figure. (B) Differential Scanning Fluorimetry (DSF) of WT and RT3a mutant MBP-tagged G2L4 RTs. Melting profiles of different protein are color-coded as in panel A. The plots show the derivative of fluorescence intensity as a function of temperature, indicating transitions or melting points indicative of structural changes or stability shifts. (C and D) Biochemical activities of WT and RT3a mutant MBP-G2L4 RTs in reaction media containing 10 mM Mg 2+ (panel C) or 10 mM Mg 2+ plus 1 mM Mn 2+ (panel D). Left panels, primer extension assays with a 50-nt 3’-inverted dT-blocked DNA template, a 5-nt DNA primer, and 32 P-labeled (red asterisk) dNTPs; middle panels, snapback DNA synthesis assays with a 5’- 32 P-labeled 50-nt DNA substrate initiated by adding dNTPs; right panels, MMEJ assays with a 5’-[ 32 P]-labeled 53-nt pre-annealed partially double-stranded DNAs with an inverted dT 3’- blocking groups on one strand and 3’ overhangs with complementary 3’ CCGG sequences on the opposite strand. The pre-annealed DNAs on the left side are denoted D1/D2, and those on the right side are denoted D1’/D2’. Primer extension reactions were initiated by adding an equimolar mix of 1 mM dNTPs (1 mM each of dATP, dCTP, dGTP and dTTP) and trace [α- 32 P]-dTTP. snapback DNA synthesis and MMEJ reactions with 5’-labeled substrates were initiated by adding an equimolar mix of 1 mM dNTPs. Reactions were incubated at 37°C for times up to 180 min, and Products (P) and substrates (S) were analyzed by electrophoresis in a denaturing polyacrylamide gel against 5’-[ 32 P]-labeled synthetic ssDNA size markers for primer extension reactions and in a non-denaturing polyacrylamide gel against 5’-[ 32 P]-labeled dsDNA size markers (Low Molecular Weight DNA Ladder; New England Biolabs) for snapback DNA synthesis and MMEJ assays. Size markers were run in parallel lanes with positions indicated to the left of the gels. The plots show the fraction of product based on the relative intensity of product and substrate bands as a function of time. The Tables to the right of the plots indicate the rate constant (k obs ) and amplitude (Ampl.) for the production of products with curves fit to a first-order rate equation to obtain average values and variance indicated by error bars for two repeats of the experiment.

Article Snippet: Products were analyzed by electrophoresis on a non-denaturing 12% polyacrylamide gel against double-stranded DNA size markers (5’-labeled Low Molecular Weight DNA Ladder; New England Biolabs) with TBE (89 mM Tris, 89 mM Borate and 2 mM EDTA) buffer.

Techniques: Size-exclusion Chromatography, Purification, Mutagenesis, Fluorescence, Labeling, DNA Synthesis, Blocking Assay, Incubation, Electrophoresis, Molecular Weight

Journal: bioRxiv

Article Title: Structural basis for the evolution of a domesticated group II intron-like reverse transcriptase to function in host cell DNA repair

doi: 10.1101/2025.01.14.632616

Figure Lengend Snippet: (A) Size-exclusion chromatography of WT G2L4 RTs with a cleaved MBP tag (left panel) and a G2L4 RT C-terminal deletion mutant (ι1401-411 amino acids; right panel). The molecular weights in parenthesis were calculated based on the elution volume of the peak (dashed line) relative to the protein standard calibration graph of Figure S1A. (B) Differential Scanning Fluorimetry (DSF) assays. Left panel, G2L4 RT with (black) or without (red) an MBP tag; middle panel, C-terminal deletion mutant of G2L4 RT (ι1401-411 amino acids, green) versus WT G2L4 RT (black); right panel, dimer interface NTE mutant (R12/Y15/R19A; red); Thumb (T) domain mutant (R397/R398/R404A; blue); and NTE+T mutant (R12/Y15/R398A; yellow). The plots show the derivative of fluorescence intensity as a function of temperature, highlighting transitions or melting points indicative of structural changes or stability shifts. (C) Coomassie blue-stained non-denaturing gel for WT and mutant G2L4 RTs with an N-terminal MBP tag. Dimer and monomer bands are labeled to the right. The fractions of monomer bands indicated below the gel were calculated by using ImageQuant TL 10.2 software. (D) Terminal transferase assays with or without Mn 2+ of MBP-tagged G2L4 RT dimer interface mutants using a 5’-labeled 50-nt DNA substrate, as described in Figure S3B.

Article Snippet: Products were analyzed by electrophoresis on a non-denaturing 12% polyacrylamide gel against double-stranded DNA size markers (5’-labeled Low Molecular Weight DNA Ladder; New England Biolabs) with TBE (89 mM Tris, 89 mM Borate and 2 mM EDTA) buffer.

Techniques: Size-exclusion Chromatography, Mutagenesis, Fluorescence, Staining, Labeling, Software