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: The DNA template (1 µM) was pre-annealed to 200 µM of a 5-nt DNA primer in 100 µL of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating to 95°C for 3 min followed by cooling to 25°C at 0.1°C /min in a T100 thermal cycler (Bio-Rad).

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: The DNA template (1 µM) was pre-annealed to 200 µM of a 5-nt DNA primer in 100 µL of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating to 95°C for 3 min followed by cooling to 25°C at 0.1°C /min in a T100 thermal cycler (Bio-Rad).

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) Wide-angle view of NTE/RT0 (red) and thumb domain (green) interactions in a GII RT complex structure with RNA template (violet), annealed DNA primer (cyan), and incoming dATP (green) shown as sticks, and a catalytic Mg 2+ shown as a green sphere. Numbers indicate nucleotide positions relative to 5’ end of the template, with -1 corresponding to the position of the templating nucleotide and incoming dATP. (B) Close-up view of interacting (dashed black line) residues of GII RT RT0 loop and the RNA template. (C) Close-up view of interacting residues of GII RT thumb domain and DNA primer. (D) Wide-angle view of the NTE/RT0 (red) and thumb domain (green) interactions in the G2L4 RT snapback DNA complex structure with the DNA template strand (violet), annealed to DNA primer strand (cyan), and incoming dCTP (yellow) depicted as sticks. Numbers indicate nucleotide positions relative to the 5’ end of the template, with N-1 corresponding to the position of the templating nucleotide and incoming dCTP. (E) Close-up view of the interacting serine residues of G2L4 RT NTE/RT0 loop and DNA template strand in the snapback DNA complex structure. A divalent metal ion is shown as a pink sphere. (F) Close-up view of the interacting residues of G2L4 RT thumb domain and DNA primer strand in the snapback DNA complex. (G and H) Biochemical assays of WT and S3/G3 mutant G2L4 RTs in reaction media containing 10 mM Mg 2+ (panel G) or 10 mM Mg 2+ plus 1 mM Mn 2+ (panel H). Left panel, primer extension assays; middle panels, snapback DNA synthesis assays; right panel, MMEJ assays. Biochemical assays were done as in . The plots show the average values and variance for two repeats of the experiment. Red asterisks in schematics at the bottom indicate an internally labeled DNA strand for primer extension assays or a 5’-labeled strand for snapback DNA synthesis and MMEJ assays.

Article Snippet: The DNA template (1 µM) was pre-annealed to 200 µM of a 5-nt DNA primer in 100 µL of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating to 95°C for 3 min followed by cooling to 25°C at 0.1°C /min in a T100 thermal cycler (Bio-Rad).

Techniques: Mutagenesis, DNA Synthesis, Labeling

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) Multiple sequence alignments of the NTE/RT0 loop region of G2L4 RT (top) with those of other RTs (NCBI accession numbers or PDB codes to the right of protein names) performed by T-Coffee via Jalview , . The color scheme of amino acids boxes is based on their Chou-Fasman two turn propensity (red, highest turn propensity; cyan, lowest turn propensity) . The numbers at the top indicate the positions of the G2L4 RT sequence. The asterisks indicate conserved serine residues in G2L4 RTs. (B) Terminal transferase assays of MBP-tagged G2L4 wild-type and S3/G3 mutant RTs in the presence of 10 mM Mg 2+ (left assays) or 10 mM Mg +2 + 1 mM Mn 2+ (right assays) using a 5’- labeled 50-nt DNA substrate, as described in Figures S3B. 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. The numbers to the left of the gels indicate the positions of 5’-labeled RiboRuler Low Range RNA Ladder size markers run in a parallel lane. The plots show the average values and variance for two repeats of the experiment. The Tables to the right of the plots indicate rate constants (k obs ) and amplitudes (Ampl.) for synthesized DNA products by WT and S3/G3 RT0 loop mutant G2L4 RTs with a curve fit 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: The DNA template (1 µM) was pre-annealed to 200 µM of a 5-nt DNA primer in 100 µL of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating to 95°C for 3 min followed by cooling to 25°C at 0.1°C /min in a T100 thermal cycler (Bio-Rad).

Techniques: Sequencing, Mutagenesis, Labeling, Incubation, Synthesized

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) Left panel, view of a G2L4 RT dimer showing interacting regions of the NTEs of monomers A and B; right panel, 90° vertically rotated view showing interacting regions of the thumb domains of monomers A and B. Regions in the left-hand monomer are color-coded as in . (B) Interactions of NTE residues R12, Y15 and R19 of monomer A with NTE residues D46, Y15 and E41 of monomer B. The NTEs of monomer A and B are shown in red and white, respectively. Black dashed lines highlight salt bridges or hydrogen bonds. (C) Interactions of thumb domain residues R397, R398, and R404 of monomer A with thumb domain residues E382, D371 and D348 of monomer B. The Thumb domains of monomer A and B are shown in light green and white, respectively. Black dashed lines highlight salt bridges or hydrogen bonds. (D) WebLogos of interacting regions of the NTEs and the thumb domains of G2L4 RT dimers. Interactions between amino acid residues in the two monomers are indicated by black dashed lines with dashed circular line above the Y15 indicating interactions between the same residue in the two monomers. Amino acids in α-helical (H) and random coil (C) regions of apoenzyme and snapback substrate bound G2L4 RTs based on the crystal structures are indicated below the amino acid sequence. (E and F) Biochemical assays of G2L4 RT dimer interface mutants in reaction media containing 10 mM Mg 2+ (panel E) or 10 mM Mg 2+ plus 1 mM Mn 2+ (panel F). Left panel, primer extension assay; middle panels, snapback DNA synthesis assays; right panel, MMEJ assays. Reactions were done as in . Ampl. values in parentheses indicate fixed amplitudes for reactions that did not reach an end point based on the average amplitude value for those that reached a clear end point during the experiment. The plots show the average values and variance for two repeats of the experiment. Red asterisks in schematics at the bottom indicate an internally labeled DNA strand for primer extension assays or a 5’-labeled strand for snapback DNA synthesis and MMEJ assays.

Article Snippet: The DNA template (1 µM) was pre-annealed to 200 µM of a 5-nt DNA primer in 100 µL of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating to 95°C for 3 min followed by cooling to 25°C at 0.1°C /min in a T100 thermal cycler (Bio-Rad).

Techniques: Residue, Sequencing, Primer Extension Assay, DNA Synthesis, Labeling

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 and B) Biochemical assays of G2L4 RT NTE mutants in reaction media containing 10 mM Mg 2+ (panel A) or 10 mM Mg 2+ plus 1 mM Mn 2+ (panel B). Left, primer extension assays; right, MMEJ assays. The plots show the average values and variance for two repeats of the experiment. 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. (D) Model of a G2L4 RT dimer with two active monomers bound to a 5-bp annealed microhomology between the single-stranded 3’ overhangs from the left side (cyan) and right side (violet) of the DSB. A dashed red circle highlights a gap between single-stranded region of the MMEJ substrate and the trailing active monomer. (E) Model of a G2L4 RT dimer with an active and inactive monomers bound to a 7-bp annealed microhomology between the same single-strand 3’ overhangs as panel C. A steric clash is highlighted in a dashed red circle. (F) Wide-angle views of the RT0 region of LINE-1 RT (PDB: 8C8J) with bound RNA template/DNA primer substrate and dTTP. The RNA template (violet), DNA primer (cyan), and bound dTTP (light blue/orange) are depicted as sticks. Tower (light yellow) and Wrist (pink) are additional regions of LINE-1 RT. (G) Wide-angle views of the RT0 region of Bombyx mori R2 element RT (PDB:8GH6) modeled with the same DNA temple/DNA primer substrate as G2L4 RT. The DNA template (violet), DNA primer (cyan), and bound dTTP (blue base) and dCTP (yellow base) are depicted as sticks.

Article Snippet: The DNA template (1 µM) was pre-annealed to 200 µM of a 5-nt DNA primer in 100 µL of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating to 95°C for 3 min followed by cooling to 25°C at 0.1°C /min in a T100 thermal cycler (Bio-Rad).

Techniques: