Fig. 4: The C-terminal tail of TOP3B stimulates DNA/RNA binding and improves the catalytic processivity of TOP3B.
From: Structural and biochemical basis for DNA and RNA catalysis by human Topoisomerase 3β
a Overall architecture of TOP3B based on AlphaFold-predicted 3D model. The conserved TOP3Bcore is colored in green and the C4-type zinc-finger motifs and an Arg-Gly/Arg-Pro-rich fragment in the C-terminal tail are indicated by arrows. b Domain structures of the full-length TOP3B, of the RG/RP-rich fragment depleted mutant and of the TOP3Bcore. Roman numerals refer to the conserved domains I-IV of typical type IA topoisomerases and numbers to amino-acid positions. c DNA cleavage and reversal assays with WT TOP3B and mutants displayed in panel b. Same molar concentrations of DNA/RNA and enzymes were tested in all the assays. Steady-state cleavage products (Clev) and high-salt reversal products (Rev) at various time points are indicated above each lane. d same as c for RNA cleavage and reversal. e, f EMSA assays using catalytic inactive TOP3B-Y336F and TOP3Bcore-Y336F showing reduced DNA and RNA binding upon C-terminal tail removal. Arrows and cartoons indicate enzyme-substrate bands. Enzyme concentrations are indicated above each lane. Each gel-based assay in c- was repeated 2-3 times independently with similar results. g Schematic illustration proposing how the zinc-finger and the RG/RP-rich fragments within the C-terminal tail stabilize the binding of TOP3B to single-stranded DNA/RNA. h Experimental setup and procedure for the magnetic-tweezers experiments. -SC, negatively supercoiled DNA. F, magnetic force. Extending force used in the assay: 0.2 pN. i Typical time trace and abstracted contour line showing stepwise removal of DNA negative supercoils within a single TOP3B relaxation burst. j Histogram showing the distribution of TOP3B step-amplitude (change of DNA linking number in each step/catalytic cycle). Fitting to a Gaussian gives a mean of 0.99 ± 0.03 (SEM; n = 59). k Representative time-traces of a typical TOP3B multi-cycle burst and a single-cycle burst of TOP3Bcore. l Distributions of TOP3B and TOP3Bcore catalytic processivity (total number of DNA supercoils removed in each relaxation burst) showing the multi-cycle DNA relaxation profile of TOP3B and the single-cycle profile of TOP3Bcore.
