Fig. 5. Unwinding assays.

a Comparison of the structure of full-length yeast Brr2 (left) and the present ASCC2^1–434-ASCC3^1–207 complex (right) after superposition of the plug/plug-like domains (blue). Human Brr2 in the U4/U6•U5 tri-snRNP or in the pre-catalytic spliceosome exhibits a plug domain very similar to yeast Brr2 blocking RNA access^42,43 (PDB IDs 3JCR, 6QW6, 6QX9). NC, N-terminal cassette; CC, C-terminal helicase cassette. b Experimental setup for stopped-flow/fluorescence-based unwinding assays. Gray sphere, helicase; star symbol, fluorophore (Alexa 488); Q, quencher (Atto 540 Q). c Stopped-flow/fluorescence-based assays monitoring unwinding of a 3’-overhang RNA by Brr2^FL or Brr2^HR, showing that auto-inhibition, which has been documented for Brr2 using gel-based unwinding assays³³, can be readily detected using the present experimental setup. d Stopped-flow/fluorescence-based assays monitoring unwinding of DNA bearing a 3’-overhang (3’-ovh), and lack of unwinding of a 5’-overhang (5’-ovh) DNA by ASCC3^HR upon addition of ATP. e Nucleotide preference of ASCC3^HR in unwinding a 3’-overhang DNA. f Stopped-flow/fluorescence-based assays monitoring unwinding of 3’-overhang DNA by ASCC3^NC compared to ASCC3^HR using ATP. g Stopped-flow/fluorescence-based assays monitoring unwinding of a 3’-overhang DNA by the indicated ASCC3 constructs in the absence or presence of ASCC2 using ATP. Insets, side-by-side presentation of the largely overlapping curves. h Stopped-flow/fluorescence-based assays monitoring unwinding of a 3’-overhang DNA by ASCC3^HR alone or in the presence of ASCC3^NTR or ASCC3^42–161 using ATP. Insets, side-by-side presentation of the largely overlapping curves. FL/HR/NC/NTR/(42–161), ASCC3 variants as defined in the text.