Extended DNA threading through a dual-engine motor module in the activating signal co-integrator complex

Abstract Activating signal co-integrator complex (ASCC) supports diverse genome maintenance and gene expression processes. Its ASCC3 subunit is an unconventional nucleic acid helicase, harboring tandem Ski2-like NTPase/helicase cassettes crucial for ASCC functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. Here, we present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as in vitro and cellular functional analyses of the ASCC3-ASC1/TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. ASC1 docks on ASCC3 via a zinc finger domain and stimulates the helicase by positioning a C-terminal ASC1-homology domain next to the C-terminal helicase cassette of ASCC3, likely assisting the DNA exit. ASC1 binds ASCC3 mutually exclusively with the DNA/RNA dealkylase, ALKBH3, directing ASCC for specific processes. Our findings define ASCC3-ASC1/TRIP4 as a tunable motor module of ASCC that encompasses two cooperating ATPase/helicase units functionally expanded by ASC1/TRIP4.