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

doi:10.21203/rs.3.rs-2007381/v1

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

https://doi.org/10.21203/rs.3.rs-2007381/v1

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published 05 Apr, 2023

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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.

Human activating signal co-integrator complex (ASCC) has been implicated in a surprisingly diverse range of genome maintenance and gene expression processes, including transcriptional regulation, DNA repair and ribosome quality control. ASCC was originally described to comprise four subunits, i.e., activating signal co-integrator 1/thyroid receptor-interacting protein 4 (ASC1/TRIP4; “ASC1” in the following), ASCC1, ASCC2 and ASCC3.¹ However, different sets of ASCC subunits have been implicated in different ASCC-dependent processes, suggesting that ASCC’s subunit composition or requirements may differ for different cellular functions. By associating with basal transcription factors², nuclear receptors^2,3 and/or various co-activators^1,2,4,5, ASCC is thought to establish distinct transcription co-activator complexes in response to different cellular conditions^2,4. Moreover, the ASCC3 subunit has been identified as a modulator of antiviral type I interferon-stimulated genes during infections by positive-strand RNA viruses.⁶ ASCC2 and, in particular, ASCC3 have also been implicated in the suppression of long mRNA isoforms, due to a decrease in transcription elongation rates and instigation of alternative last exon splicing, upon UV irradiation or exposure to agents that give rise to bulky DNA lesions; a short ascc3 transcript, itself originating from alternative last exon splicing, in turn acts as a long non-coding RNA during transcriptional recovery.^7,8

ASCC3, supported by ASC1, ASCC1 and ASCC2, is also involved in ribosome and translation quality control pathways.^9–12 In contrast, only ASCC1, ASCC2 and ASCC3, have additionally been found to be important for DNA alkylation damage repair^13–15, for which the factors associate with the single-stranded (ss) DNA/RNA-specific α-ketoglutarate/iron-dependent dioxygenase, ALKBH3;^16,17 ASC1 has not yet been implicated in DNA alkylation damage repair. Finally, ASCC, possibly in different constellations, may also help mediate RNA modification/repair processes. For example, a proteomics analysis suggested that ASC1, ASCC1, ASCC2 and ASCC3 interact with ZCCHC4, a methyl-transferase that introduces a m⁶A modification at position A4220 of 28S rRNA.¹⁸ Furthermore, ASCC3 is required for efficient, ALKBH3-dependent removal of m¹A and m³C modifications from mRNAs, and for alkylation-induced P-body formation.¹⁹

ASCC3 contributes to all of the above ASCC-related functions. It is a large nucleic acid-dependent NTPase that can act as a 3’-to-5’ translocase/helicase.^13,20 NTPase-fueled remodeling of nucleic acids or nucleic acid-protein complexes by ASCC3, therefore, likely constitute central activities for all of ASCC’s diverse cellular roles. For example, during DNA alkylation damage repair, ASCC3 generates single-stranded DNA for dealkylation by ALKBH3.^13,14 Furthermore, mutations in conserved NTPase/helicase motifs of ASCC3 interfere with ASCC-mediated splitting of stalled ribosomes during ribosome or translation quality control^9–12, and ASCC3 has been suggested to disassemble ribosomes collided on alkylated mRNAs for dealkylation by ALKBH3¹⁹.

ASCC3 is an unconventional nucleic acid-dependent NTPase that is closely related to the spliceosomal RNA helicase, U5 small nuclear ribonucleoprotein 200 kDa (SNRNP200/BRR2). ASCC3 and SNRNP200 contain a tandem array of Ski2-like helicase cassettes (N-terminal cassette, NC; C-terminal cassette, CC), preceded by ~ 400-residue N-terminal regions that can auto-inhibit the helicase activities.^20–22 In SNRNP200, only the NC is an active NTPase and helicase, while the CC acts as an intra-molecular helicase co-factor.^21,23 In contrast, both helicase cassettes in ASCC3 may be enzymatically active.^12,13,20 However, presently the molecular mechanisms underlying ASCC3 nucleic acid translocase/helicase activities and its regulation are poorly understood.

Here, we find a hitherto unobserved mechanism of nucleic acid translocation/unwinding in ASCC3 and reveal that it is regulated by ASC1. Using cryogenic electron microscopy (cryoEM)/single-particle analysis (SPA) and DNA-protein cross-linking/mass spectrometry (CLMS)-based structural analyses as well as systematic protein interaction, DNA binding and unwinding assays, we show that ASCC3 can thread DNA through both of its helicase cassettes. ASC1 docks to the ASCC3 CC via a zinc finger (ZnF) domain, positioning its ASC1-homology (ASCH) domain such that it can engage DNA exiting from ASCC3. We also present evidence that ASC1 and ALKBH3 engage ASCC3 in a mutually exclusive manner and that ASC1 does not affect ASCC-dependent DNA alkylation damage repair, suggesting that ASC1 and ALKBH3 are facultative, process-specific ASCC subunits or auxiliary proteins.

ASC1 directly associates with the helicase region of ASCC3

ASCC1 and ASCC2 directly interact with ASCC3,^11,15,20 suggesting that ASCC3 forms the main scaffold for the ASCC and possibly an interaction platform for ASCC-auxiliary proteins. We therefore tested whether ASC1 also directly binds ASCC3 in vitro. While ASC1 did not stably interact with the ASCC3 N-terminal region (ASCC3^NTR, residues 1-400), it co-eluted with the helicase region of ASCC3 (ASCC3^HR, residues 401–2202) in analytical size-exclusion chromatography (SEC; Fig. 1a,b)

Next, we reconstituted an ASCC3^HR-ASC1 complex and determined its atomic structure via cryoEM/SPA at a nominal resolution of 3.4 Å (Fig. 2a; Supplementary Fig. 1; Supplementary Fig. 2; Supplementary Table 1). In the cryoEM reconstruction, we could trace residues 401–2183 of ASCC3^HR as well as residues 168–219 and 375–580 of ASC1 (Fig. 2b,c), capitalizing on AlphaFold-predicted models²⁴. ASCC3^HR adopts a structure very similar to the helicase region of SNRNP200 (root mean square deviation [rmsd] of 3.1 Å for 1,504 pairs of Cα atoms compared to isolated SNRNP200^HR; PDB ID 4F91; Supplementary Fig. 3)²¹. Like SNRNP200, both ASCC3 helicase cassettes contain consecutive dual RecA-like (RecA1, RecA2), winged-helix (WH), helical bundle (HB), helix-loop-helix (HLH) and immunoglobulin-like (IG) domains and associate to form a compact helicase region (Fig. 2c). An extended, irregularly structured linker (residue 1296–1306) connects the IG domain of the NC to the RecA1 domain of the CC, running closely along the body of the ASCC3 CC (Fig. 2a-c).

ASC1 exclusively associates with the CC of ASCC3^HR (Fig. 2a,b). Residues 168–219 of ASC1 fold into a dual-ZnF domain, with residues C171/C173/H178/C192 and C184/C187/C200/C203 each coordinating a zinc ion (Fig. 2b). The ZnF domain of ASC1 rests on top of the RecA1 domain of the ASCC3 CC, neighboring the extended linker to the NC (Fig. 2b) and spanning ~ 757 Å² of interface area, with hydrophobic interactions in the center and hydrophilic interactions at the periphery (Fig. 2d, left). ASC1 residues 375–424 lack a globular fold and regular secondary structure elements, except for a short helical region in residues 398–405. They form a lasso-like structure around a protruding edge of the C-terminal ASCC3 WH domain (Fig. 2b; Fig. 2d, middle), with residues 411–424 inserted deeply into a groove between the RecA1, WH, HB and IG domains of the ASCC3^HR CC, spanning ~ 1,914 Å² of interface area with ASCC3^HR. ASC1 residues 411–424 form a support for the C-terminal ASCH domain of ASC1 (residues 425–578) that further interconnects the C-terminal ASCC3 RecA1, WH and IG domains (Fig. 2b; Fig. 2d, right), spanning an additional ~ 1,321 Å² of interface area with ASCC3^HR.

The ZnF domain is required for stable docking of ASC1 on ASCC3 ^HR in vitro

Based on the structure, we designed various ASC1 fragments to probe the importance of different regions for stable complex formation with ASCC3^HR. Consistent with the cryoEM structure, the N-terminal 80 residues of ASC1 did not sustain a stable interaction with ASCC3^HR (Fig. 1c), while ASC1 residues 152–581, encompassing the ZnF domain, the lasso-like peptide and the ASCH domain, co-migrated with ASCC3^HR in analytical SEC (Fig. 1d). An N-terminal ASC1 region including the ZnF domain (residues 1-230) or the ZnF domain alone (residues 152–230) also stably bound ASCC3^HR (Fig. 1e). In contrast, C-terminal ASC1 residues 281–403, 403–581 or 281–581, containing the lasso-like peptide, the ASCH domain or both, did not support stable complex formation with ASCC3^HR (Fig. 1f), although these regions span a considerably larger interface with ASCC3^HR than the ZnF domain (see above). Thus, only the ZnF domain of ASC1 is required for stable complex formation in vitro, and only upon anchoring via the ZnF domain, the C-terminal ASCH domain and the preceding peptide region of ASC1 are stably docked on the ASCC3 CC.

Cellular interaction tests corroborate in vitro interaction patterns

To test the importance of ASC1 regions for the interaction with ASCC3 and other ASCC subunits in cells, we generated stably transfected Flp-In™ T-REx™ 293 cell lines for the inducible expression of N- or C-terminally Flag-tagged versions of full-length ASC1 or truncation variants lacking either N-terminal regions including the ZnF domain (ASC1^Δ1− 276) or lacking the C-terminal ASCH domain (ASC1^{Δ403− 581}). Immuno-fluorescence microscopy showed that all constructs were located to both the cytosol and the nucleus (Fig. 3a). We then immuno-precipitated the Flag-tagged ASC1 variants with α-Flag antibodies and probed the eluates for the presence of other ASCC subunits by western blot. Irrespective of the position of the tag, ASC1 and ASC1^{Δ403− 581} (lacking the ASCH domain) co-precipitated ASCC1, ASCC2 and ASCC3 (Fig. 3b). In contrast, no interaction with these ASCC subunits was detected by co-precipitation with ASC1^Δ1− 276 (lacking the ZnF domain; Fig. 3b).

To further test the relevance of ASCC3^HR-ASC1 contacts observed in our cryoEM structure for the interaction of ASCC3 and ASC1 in cells, we transfected 293T cells for the expression of N-terminally HA-tagged versions of ASC1. In these ASC1 variants, either the ZnF domain was precisely deleted (ΔZnF; deletion of residues 168–219), three residues that engage in hydrophobic interactions with ASCC3^HR were exchanged for alanines (LLI-AAA, ASC1^{L174A − L180A−I190A}; Fig. 2d, left) or two cysteines coordinating the first (C171) and second (C184) Zn²⁺ ion were exchanged for alanines (CC-AA, ASC1^{C171A − C184A}). While wild type (wt) ASC1 efficiently co-immuno-precipitated endogenous ASCC3, the ΔZnF and CC-AA variants entirely lost the ability to immuno-precipitate ASCC3, and the ASCC3 interaction of the LLI-AAA variant was strongly reduced (Fig. 3c).

Together, the results of these cellular interaction studies are fully in line with the in vitro ASCC3^HR-ASC1 interaction profiles. They confirm that the ZnF domain of ASC1 is the main ASCC3-interacting domain of ASC1, via which ASC1 also seems to be incorporated into the ASCC, and suggest that ASC1, ASCC1 and ASCC2 can concomitantly interact with ASCC3. The observations also confirm that our cryoEM structure closely represents the mode of interaction of ASCC3 and ASC1 in cells.

ASC1 activates ASCC3 helicase activity without influencing ASCC3 ATPase activity

To test the effect of ASC1 on the helicase activity of ASCC3^HR, we conducted fluorescence-based unwinding assays in a stopped-flow device (Fig. 4a). As this assay tested multiple rounds of unwinding, the observed time traces were fit to a double exponential equation, and amplitude-weighted unwinding rate constants (k_uaw) were calculated for the comparison of unwinding efficiencies.^25–27 ASCC3^HR alone efficiently unwound the substrate DNA (k_uaw = 0.024 s^− 1), but unwinding was further stimulated 2.3-fold by ASC1 (k_uaw = 0.054 s^− 1; Fig. 4b; Supplementary Table 2). In contrast, both ASC1^1–230 (encompassing the ZnF domain), which stably bound ASCC3^HR in analytical SEC, as well as ASC1^403–581 (encompassing the ASCH domain and preceding peptide), which did not co-migrate with ASCC3^HR in analytical SEC, only marginally affected the ASCC3^HR helicase activity (k_uaw = 0.030 s^− 1 and 0.035 s^− 1, respectively; Fig. 4b; Supplementary Table. 2). Thus, while the ASC1 ZnF domain alone can stably bind to ASCC3^HR, it does not efficiently activate ASCC3^HR helicase activity, for which the lasso-like peptide and ASCH domain are also required.

Next, we asked which helicase cassette of ASCC3^HR is preferentially regulated by ASC1. To this end, we employed ASCC3^HR variants, in which a crucial motif II aspartate of the NC (D611) or CC (D1453) was exchanged for an alanine (Fig. 4c), abrogating NTPase/helicase activity in the respective cassette.²⁰ ASCC3^HR,D1453A, bearing an inactive CC, unwound DNA at a reduced rate (k_uaw = 0.011 s^− 1), while the unwinding activity of ASCC3^HR,D611A, containing an inactive NC, was strongly reduced (k_uaw n.d.; Fig. 4d), suggesting that both cassettes are required for full ASCC3 helicase activity. Only the construct bearing an inactive CC was stimulated by ASC1 to quantifiable levels (ASCC3^HR,D1453A-ASC1 k_unw = 0.024 s^− 1; Fig. 4d; Supplementary Table 2).

NTPase activity associated with both ASCC3^HR cassettes was further corroborated by DNA-stimulated ATPase assays. ASCC3^HR,D1453A (inactive CC) and ASCC3^HR,D611A (inactive NC) exhibited ~ 28 and ~ 73% of the DNA-stimulated ATPase activity of wt ASCC3^HR, while the DNA-stimulated ATPase activity of the ASCC3^{HR,DD611/1453AA} variant, with motif II changes in both cassettes, was negligible (Fig. 4e; Supplementary Fig. 4). As expected if the implemented residue exchanges selectively abrogated ATPase activity in the respective cassette, the ATPase activity of ASCC3^HR,D1453A (inactive CC) closely matched the ATPase activity of the isolated wt NC (Fig. 4e; Supplementary Fig. 4) As we failed to produce the ASCC3 CC in isolation, a similar comparison could not be drawn between ASCC3^HR,D611A (inactive NC) and isolated wt CC. Irrespectively, in contrast to the helicase activity, the stimulated ATPase activity of ASCC3^HR was not further enhanced by ASC1 (Fig. 4e). Thus, ASC1 activates ASCC3^HR helicase activity without affecting its ATPase activity.

DNA can be threaded through both ASCC3 helicase cassettes and along ASC1

We failed to obtain cryoEM structures of ASCC3^HR or ASCC3^HR-ASC1 in complex with ssDNA or with dsDNA bearing a 3’-ss overhang. Modeling of putative ssDNA binding to the NC and CC of ASCC3^HR by superimposing a structure of the Hel308 DNA helicase in complex with DNA (PDB ID 2P6R)²⁸ on both ASCC3^HR cassettes indicated that ssDNA could be threaded consecutively through both helicase cassettes and might exit the CC close to the ASC1 ASCH domain (Fig. 5a). Positive electrostatic surface potential is in agreement with the modeled path of ssDNA, in particular for the ASCC3^HR NC (Fig. 5a). The model suggested that a minimum of 24 nucleotides (nts) of ssDNA are required to traverse the two cassettes and ASC1. In contrast, lateral entry of ssDNA to the CC, circumventing the NC, is blocked in the conformation of ASCC3^HR observed in our cryoEM structure. A requirement for DNA to enter the CC via the preceding NC would be consistent with the larger effect on helicase activity we observed upon inactivating the NC alone as compared to a ASCC3^HR variant containing only an inactive CC (see Fig. 4).

To test if, during unwinding, ASCC3^HR and ASCC3^HR-ASC1 might thread single-stranded DNA through both helicase cassettes, and in the latter case along the ASC1 ASCH domain, we conducted ultra-violet (UV) irradiation-induced cross-linking of ASCC3^HR and ASCC3^HR-ASC1 to variable-length, single-stranded oligo-T DNAs (T₁₂, T₂₄, T₃₆, T₄₈; Fig. 5b). Both ASCC3^HR and ASCC3^HR-ASC1 did not efficiently cross-link to T₁₂ ssDNA and showed stepwise increased cross-linking to T₂₄, T₃₆ and T₄₈ DNAs (cross-link efficiencies of ~ 30%, 80% and 90%, respectively; Fig. 5b,c). ASC1 alone did not efficiently cross-link to any of the DNA samples. These observations are consistent with the notion that a ssDNA region sufficiently long to traverse both cassettes is required for DNA to be efficiently engaged by ASCC3^HR or ASCC3^HR-ASC1.

Next, we subjected ASCC3^HR or ASCC3^HR-ASC1 after UV-induced cross-linking to T₄₈ ssDNA to DNase/protease digestion followed by mass spectrometric analysis of cross-linked peptide-DNA conjugates. We observed one cross-linked peptide each in ASC1 (region connecting ZnF and lasso), the RecA1 domain of the ASCC3^HR NC (corresponding to helicase motif Ia), the N-terminal WH domain and the C-terminal WH domain, as well as two cross-linked peptides in the CC IG domain (Table 1). With exception of the ASC1 peptide, we could identify one or two specific cross-linked residues in these peptides (Table 1; RecA1^NC, M546; WH^NC, Y988; WH^CC, Y1821 and Y1822; IG^CC, C2101 and Y2135). The cross-linked residues and the modeled cross-linked ASC1 peptide are positioned closely along the path of the modeled DNA (Fig. 5d). Together, these observations are consistent with the idea that during unwinding, ssDNA is threaded through both helicase cassettes and along ASC1 in the vicinity of the ASCH domain. It is, however, also possible that ASCC3^HR may undergo conformational changes upon binding to ssDNA of sufficient length, so that the substrate can engage the NC and CC independently.

Table 1: DNA-protein cross-links identified by MS.

Cross-linked pepide¹	Cross-linked residue	Trial	Domain or region	Motif²
ASC1
257-SGLEK-261	n.i.³	1	ZnF-lasso linker	-
ASCC3^HR
541-ALAAEMTDYFSR-552	M546	2	RecA1^NC	Ia
984-TASHYYIK-991	Y988	1,2	WH^NC	-
1818-IASYYYLK-1825	Y1821	2	WH^CC	-
1818-IASYYYLK-1825	Y1822	2	WH^CC	-
2096-GKPESCAVTPR-2106	C2101	2	IG^CC	-
2133-VGYIR-2137	Y2135	1,2	IG^CC	-

¹ Cross-linked residue(s) colored as in (c) and underlined

² NC helicase motif Ia, residues 536-546

³n.i., not identified

ASC1 and ALKBH3 support ASCC core subunits in distinct cellular functions

Present data suggest that ASCC core subunits may associate with different auxiliary proteins to participate in distinct genome maintenance and gene expression processes. More specifically, ASC1 has so far been found associated with ASCC-dependent transcription regulation^1,2,4,5 and ribosome quality control^9,11,12, while ALKBH3 is associated with ASCC3 during DNA dealkylation repair^13,14. We therefore wondered whether ASC1 and ALKBH3 might bind ASCC3 in a mutually exclusive manner. To test this notion, we conducted competitive SEC-based interaction studies. ASC1 and ALKBH3 did not co-migrate during SEC (Fig. 6a). A portion of ALKBH3 stably associated with ASCC3^HR in SEC, but failed to be incorporated into a pre-formed ASCC3^HR-ASC1 complex (Fig. 6a). These findings suggest that ASC1 and ALKBH3 engage ASCC3^HR in a mutually exclusive manner, possibly by taking advantage of overlapping binding sites, and that ASC1 might associate more strongly with ASCC3^HR than ALKBH3.

To further test the idea that either ASC1 or ALKBH3 associates with ASCC core subunits depending on the particular ASCC-dependent cellular process, we explored the effect of ASC1 on DNA dealkylation damage repair, where ALKBH3 is known to be involved. To this end, we knocked out ASC1 via CRISPR/Cas9-based genome engineering in human PC-3 cells (Fig. 6b) and tested the response of the edited and parental cells to methyl methanesulfonate (MMS) treatment. ASC1 knockout (KO) did not impact cell survival in the presence of even high concentrations of MMS (Fig. 6c), suggesting that ASC1 may not be involved in ASCC3/ALKBH3-mediated DNA dealkylation¹³. Together, these observations suggest that ASC1 represents a process-specific ASCC subunit that regulates ASCC3 helicase activity during ASCC-dependent transcriptional events and ribosome rescue, but may be replaced by ALKBH3 during ASCC-dependent DNA dealkylation damage repair.

ASCC is a multi-functional complex. While apparently different sets of ASCC core and auxiliary factors participate in different ASCC-dependent processes, the large nucleic acid helicase, ASCC3, seems to provide crucial molecular motor activity for all of ASCC’s multiple functions. ASCC3 has striking homology to the spliceosomal RNA helicase, SNRNP200, and the two proteins represent the only known human members of a unique sub-family of Ski2-like helicases that possess tandem helicase cassettes. In SNRNP200, only the NC is an active ATPase/RNA helicase, while the CC acts as an intra-molecular modulator of the NC helicase.^21,29

Here, we show by cryoEM-based structural analysis that ASCC3 indeed contains a dual-cassette helicase region that closely resembles the analogous region of SNRNP200, at least in the absence of factors other than ASC1. In line with previous observations^{9–11, 13,20}, our systematic DNA unwinding and ATPase assays strongly suggest that, in contrast to SNRNP200, both ASCC3 cassettes are active ATPases and helicases. Our DNA-protein CLMS analyses are consistent with a model in which ASCC3 translocates relative to ssDNA during DNA unwinding, threading one DNA strand consecutively through both helicase units. In principle, our data would also be consistent with the two ASCC3 helicase cassettes unwinding DNA independently of each other. However, in the ASCC3^HR conformation observed here, direct accommodation of ssDNA at the CC is blocked by the NC. Thus, for the latter scenario, ASCC3 would have to undergo a large conformational rearrangement that leads to a separation of its helicase cassettes if ssDNA were to be captured by the CC without being first threaded through the NC. As ASCC3 interacts with different substrate complexes and auxiliary proteins in different functional contexts, which could provoke conformational changes in ASCC3, it is conceivable that in certain scenarios the helicase activity of either individual cassette is employed, while in others the two helicase cassettes operate in tandem. Furthermore, in a given functional scenario the two cassettes may even translocate the same or different nucleic acid molecules (see also below).

The CC of SNRNP200 serves as an interaction platform for numerous proteins, several of which inhibit its NC helicase activity from a distance^30–32. In contrast, the C-terminal Jab1 domain of the large spliceosomal PRPF8 scaffold that can activate the SNRNP200 helicase directly binds the active NC.^33,34 Here, we find that similar to the situation in SNRNP200, the ASCC3 CC serves as a binding platform for the ASC1 protein. ASC1 predominantly latches onto ASCC3 via its ZnF domain, allowing the positioning of an ASCH domain close to the presumed DNA exit of the ASCC3 CC with the help of the intervening lasso peptide. However, unlike many proteins that bind the SNRNP200 CC, we show that ASC1 stimulates ASCC3 helicase activity. The ZnF docking domain is insufficient for helicase stimulation, which also requires C-terminal ASC1 regions including the ASCH domain. While we cannot yet pinpoint the precise molecular mechanism, by which ASC1 stimulates ASCC3, our DNA-protein CLMS data support the notion that the ASCH domain or neighboring regions may facilitate DNA exit from the ASCC3 CC. Indeed, the ASCH domain belongs to a large family of domains that bind nucleic acids.^35,36

Cooperation between both helicase cassettes and activation of ASCC3 helicase activity by ASC1 may be required to unfold sufficiently strong or appropriately coordinated motor activity during transcription regulatory processes and ribosome quality control, where both ASCC3 and ASC1 are involved. While the targets of ASCC3’s motor activity during transcriptional regulation are presently unknown, during ribosome quality control, ASCC3’s ATP-dependent motor activity is essential for the disassembly of the lead ribosome in collided di-somes or polysomes into ribosomal subunits.^9,11,12 As no DNA is involved in this process, ASCC3 most likely operates by engaging and translocating mRNA or rRNA regions. Indeed, we know that ASCC3 can also unwind RNA duplexes in vitro, suggesting that it is also an RNA translocase, but its RNA helicase/translocase activity is much less efficient than its DNA helicase/translocase activity (unpublished). Our analyses show that inactivation of either ASCC3 cassette leads to partial loss of ASCC3 helicase activity. Thus, splitting of ribosomes by translocating on a sub-optimal mRNA or rRNA substrate may require (a) ASCC3 resorting to a translocation mode that involves both active cassettes on the same or on different RNA molecules, (b) additional stimulation by ASC1 and/or (c) stimulation by another accessory factor that promotes ASCC3 RNA translocase/helicase function.

Recent cryoEM structures of yeast ribosome quality control trigger complex (RQT)-ribosome complexes revealed that prior to ribosome splitting the yeast ASCC3 ortholog, Slh1p, can adopt a more open conformation with fewer direct interactions between the two helicase cassettes as observed in our human ASCC3-ASC1 complex structure.³⁷ While in the imaged conformations both Slh1p helicase cassettes are potentially accessible to an RNA substrate, no corresponding substrate density was observed at either Slh1p cassette.³⁷ In the observed conformations, mRNA could apparently be accommodated directly at the Slh1p CC, but an Slh1p variant harboring an ATPase-deficient NC (Slh1p^K361R) was required to capture RQT-ribosome complexes at a stage preceding ribosome splitting³⁷, indicating that the NC ATPase/helicase activity is also required for the splitting reaction. Thus, whether both cassettes or only one of them translocate mRNA or whether one cassette engages mRNA while the other operates on an rRNA region during ribosome splitting remains to be elucidated.

Findings reported here also underscore the notion that ASCC exhibits compositional dynamics that allow it to participate in different processes. We find that ASC1, which collaborates with ASCC3 during transcriptional and ribosome quality control, binds to ASCC3 in a manner that is mutually exclusive to ALKBH3, which capitalizes on the ASCC3 helicase activity during DNA alkylation damage repair. Consistent with the idea of these two factors associating with ASCC3 in different functional scenarios, we also show that ASC1 does not impact cell sensitivity to an alkylating agent, unlike ALKBH3 or other subunits of the ASCC complex^13,15. As ASC1 seems to associate more stably with ASCC3^HR than ALKBH3, it remains to be seen if additional factors may aid ALKBH3 in displacing ASC1 for DNA dealkylation damage repair. Additional interactors may favor a conformation of ASCC3 that exhibits altered ALKBH3 affinity. It is also possible, that the protein interactions of ASCC3 may be dynamically regulated by specific post-translational modifications or by the recruitment of subsets of factors to specific sub-cellular compartments. Both of the latter principles have been shown to play a role during ASCC-related cellular processes.^{9,10,14,15,19,38,39}

Molecular cloning

DNA fragments encoding ASCC3^HR (wt, D611A, D1453A or D611A-D1453A) or ASCC3^NC were cloned into a pFL vector for expression as N-terminally His₁₀-tagged, TEV-cleavable proteins via recombinant baculoviruses in insect cells as described previously.²⁰ A DNA fragment encoding full-length (FL) ASC1 was PCR-amplified from a synthetic gene (IDT; Supplementary Table 3) and inserted into the pETM-11 or pIDS vectors (EMBL, Heidelberg) for expression as an N-terminally His₆-tagged, TEV-cleavable protein. See Supplementary Table 4 for PCR primers used. The pIDS-asc1^FL construct was Cre-recombined with pFL-ascc3^HR for co-expression via a recombinant baculovirus in insect cells. DNA fragments encoding ASC1^1–80, ASC1^1–230, ASC1^152–230, ASC1^152–581, ASC1^281–403, ASC-1^281–581 or ASC-1^403–581 were amplified via PCR from the pETM-11-asc1^FL, and re-cloned into the pETM-11 vector. A DNA fragment encoding full-length ALKBH3 was PCR-amplified from a cDNA library of human HeLa cells and inserted into the pETM-11 vector for expression as an N-terminally His₆-tagged, TEV-cleavable protein. All constructs were verified by Sanger sequencing.

For the preparation of an ASC1 sgRNA vector, we followed a previously established method⁴⁰, cloning the target sequence into the pLenti-CRISPRV2 vector⁴¹. Primers used for generating the DNA fragment containing the target sequence is shown in Supplementary Table 4.

For expression of HA-tagged ASC1 variants, DNA fragments encoding wt or ΔZnF ASC1 were cloned into pENTR-3C using a synthetic gene (IDT; Supplementary Table 3). Vectors encoding HA-tagged variants ASC1^{L174A − L180A−I190A} or ASC1^{C171A − C184A} were created using the In-Fusion Snap Assembly mutagenesis kit (Takara Bio #683945). Each construct was then cloned into pHAGE-HA-Blast vector¹⁴ via Gateway recombination. All constructs were verified by Sanger sequencing.

Generation of cell lines

Stably transfected Flp-In™ T-REx™ 293 cell lines for the tetracycline-inducible expression of ASC1 variants with N-terminal 2xFlag-His₆ or C-terminal His₆-2xFlag tags were generated according to the manufacturer’s guidelines.²⁰ Transfection of the parental cell line was done using X-tremeGENE HP DNA Transfection Reagent (Sigma Aldrich). After hygromycin-based selection of cells that had genomically integrated the expression cassette, tetracycline-induced expression of the tagged proteins was confirmed by western blotting using a monoclonal α-Flag M2 antibody (Sigma Aldrich #F3165; 1:7500). For expression of HA-tagged ASC1 variants, the pHAGE-HA-ASC1 vectors encodin HA-tagged ASC1^wt, ASC1^ΔZnF, ASC1^{L174A − L180A−I190A} or ASC1^{C171A − C184A}, were transfected into 293T cells using Transit293 transfection reagent (Mirus Bio).

CRISPR/Cas9-based genome editing

The ASC1 sgRNA expression vector was transfected into the Lenti-X 293T cell line (Takara Bio) together with psPAX2 and pCMV-VSVG (Addgene) for lentivirus production. The virus-containing culture medium was collected 72 h post-transfection. Human PC-3 cells were infected with the viral medium and individual clones were selected in 96-well plates. The single KO colonies were analyzed by western blot using an α-ASC1 antibody (sc-376916, Santa Cruz).

Recombinant protein production and purification

ASCC3^HR variants and ASCC3^NC were produced in High Five cells as described previously.²⁰ Cell pellets were re-suspended in 20 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 10 mM imidazole, 1 mM DTT, 8.6% (v/v) glycerol (lysis buffer 1), supplemented with cOmplete™ protease inhibitors (Roche) and lysed by sonication using a Sonopuls Ultrasonic Homogenizer HD (Bandelin). The lysate was cleared by centrifugation and filtration. The protein of interest (POI) was captured on Ni²⁺-NTA resin in a gravity flow column, washed with lysis buffer 1 and eluted with lysis buffer 1 containing 400 mM imidazole. Fractions enriched for the POI were supplemented with 1/10 (w/w) TEV protease and dialyzed against 20 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 1 mM DTT, 8.6% (v/v) glycerol (dialysis buffer) overnight. The sample was then diluted to 100 mM NaCl and loaded onto a HiTrap Heparin HP column (Cytiva), pre-equilibrated with lysis buffer 1 containing 100 mM NaCl. After washing with lysis buffer 1 containing 100 mM NaCl, the POI was eluted with a linear gradient to lysis buffer 1 containing 1.5 M NaCl. The fractions containing the POI were pooled and concentrated with a centrifugal concentrator (100 kDa molecular mass cut-off). The concentrated sample was further purified by SEC on a Superdex 200 10/300 GL column (Cytiva) in 20 mM HEPES-NaOH, pH 7.5, 250 mM NaCl, 5% (v/v) glycerol, 1 mM DTT (SEC buffer). Fractions containing the POI were combined, concentrated, aliquoted, flash-frozen in liquid nitrogen and stored at -80°C.

For preparation of the ASCC3^HR-ASC1^FL complex, ASC1^FL was co-produced with ASCC3^HR in High Five cells. Cell pellets were re-suspended in lysis buffer 1 supplemented with cOmplete™ protease inhibitors, 1 mM DTT and 20 mM imidazole. The samples were lysed by sonication, then the suspension was centrifuged at 56,000 x g for 1 h, the soluble extract was further filtered through 0.8 µM pore size membrane filters (Millipore). The filtered fractions were collected and incubated with Ni²⁺-NTA resin pre-equilibrate with lysis buffer 1 for 2 h with gentle rotation at 4°C. POI-bound resin was loaded on a gravity flow column, washed with lysis buffer 1 and the POI was eluted with lysis buffer 1 containing 400 mM imidazole. To remove the His_6/10-tags, 1/10 (w/w) of TEV protease was added and the sample and dialyzed against dialysis buffer overnight. Subsequently, the sample was diluted to 50 mM NaCl and loaded on a 5 ml StrepTrap HP column (Cytiva) pre-equilibrated with lysis buffer 1 containing 50 mM NaCl. After washing with lysis buffer 1 containing 50 mM NaCl, the POI was eluted in a linear gradient to lysis buffer 1 containing 1.5 M NaCl. Fractions containing the POI were combined, diluted to 50 mM NaCl, loaded on a 5 ml HiTrap Heparin HP column, washed and eluted in a linear gradient with lysis buffer 1 containing 1.5 M NaCl. Fractions containing the POI were pooled, concentrated and further purified by SEC on a Superdex 200 10/600 GL column (Cytiva) in 20 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 1 mM DTT. Fractions containing the POI were combined, concentrated, aliquoted, flash-frozen in liquid nitrogen and stored at -80°C.

For production of isolated ASC1 variants, the corresponding pETM-11 vectors were transformed into Escherichia coli BL21 (DE3) cells by electroporation for protein production via auto-induction at 18°C.⁴² Cells were harvested when cultures reached an optical density (600 nm) of 10. Cell pellets were re-suspended in lysis buffer 1 and supplemented with cOmplete™ protease inhibitors. After sonication, the lysate was cleared by centrifugation. The POI was captured on Ni²⁺-NTA resin in a gravity flow column, washed with lysis buffer 1 and eluted with lysis buffer 1 containing 400 mM imidazole. Fractions enriched for the POI were supplemented with 1/10 (w/w) TEV protease and dialyzed against dialysis buffer overnight. Dialyzed samples were passed through a Ni²⁺-NTA gravity flow column to remove the cleaved His₆-tag and TEV. For ASC1^FL, ASC1^152–581, ASC1^281–581 and ASC1^403–581 fragments, the samples were diluted to 100 mM NaCl, loaded on a HiTrap Heparin HP column, washed and eluted in a linear gradient to lysis buffer 1 containing 1.5 M NaCl. Fractions containing the POI were combined, concentrated and further purified on a Superdex 200 16/600 GL column in SEC buffer.

For purification of the ASC1^1–80, ASC1^1–230, ASC1^152–230 and ASC1^281–403 fragments, the Heparin column step was omitted and the final gel filtration was conducted in 20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 1 mM DTT on a HiLoad 16/60 Superdex 75 pg column (Cytiva).

For production of ALKBH3, the corresponding pETM-11 vector was transformed into E. coli C2566 cells by electroporation for protein production via IPTG induction at 37°C. Cell pellets were re-suspended in 20 mM TRIS-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole, 1 mM DTT, 0.1 mM PMSF (lysis buffer 2), and lysed by sonication. The lysate was cleared by centrifugation. The supernatant was loaded onto a Ni²⁺-NTA column, washed with lysis buffer 2 and the POI was eluted with a linear gradient to lysis buffer 2 containing 400 mM imidazole. Fractions enriched for the POI were combined, supplemented with 1/20 (w/w) TEV protease and dialyzed against dialysis buffer overnight. The sample was then diluted to 100 mM NaCl and loaded onto a HiTrap Heparin HP 5 ml column (Cytiva), pre-equilibrated with dialysis buffer containing 100 mM NaCl. After washing with dialysis buffer containing 100 mM NaCl, the POI was eluted with a linear gradient to dialysis buffer containing 1.5 M NaCl. The fractions containing the POI were pooled and concentrated with a centrifugal concentrator (10 kDa molecular mass cut-off). The concentrated sample was further purified by SEC on a Superdex 75 10/60 GL column (Cytiva) in 20 mM TRIS-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT. Fractions containing the POI were combined, concentrated, aliquoted, flash-frozen in liquid nitrogen and stored at -80°C.

Analytical size exclusion chromatography

Analytical SEC-based interaction tests were conducted in 20 mM HEPES-NaOH, pH 7.5, 250 mM NaCl, 5% (v/v) glycerol, 1 mM DTT. 100 pmol of ASCC3^HR were mixed with other proteins in a two to ten-fold molar excess in a final reaction volume of 80 µl. After incubation of the mixtures on ice for 30 min, the samples were loaded on a Superdex 200 3.2/300 analytical size exclusion column (Cytiva). 50 µl fractions were collected and subjected to SDS-PAGE analysis. Protein bands were visualized by Coomassie staining except for gels containing ASC1^1–80 or ASC1^152–230, which were imaged by silver staining.

For testing competitive binding of ASC1 and ALKBH3 to ASCC3^HR, 120 pmol of ASCC3^HR (or of pre-formed ASCC3^HR-ASC1 complex) were mixed with 360 pmol each of ASC1 and ALKBH3 (or of ALKBH3) in a volume of 100 µl. After 30 min of incubation on ice, the samples were loaded on a Superdex 200 3.2/300 analytical size exclusion column. 50 µl fractions were collected and subjected to SDS-PAGE analysis. The proteins were visualized by Coomassie staining.

DNA unwinding assays

DNA duplex unwinding activity was assessed in fluorescence-based stopped-flow experiments on a SX-20MV spectrometer (Applied Photophysics).^26,27 The DNA substrate contained a 12-base pair duplex region and a 31-nucleotide 3’-ss overhangs, with an Alexa 488 fluorophore on the short strand and an Atto 540 Q quencher on the complementary strand ([(Atto 540 Q]5’-GGCCGCGAGCCGGAAATTTAATTATAAACCAGACCGTCTCCTC-3’; 5’-CGGCTCGCGGCC-3’[Alexa 488]; duplex region in bold). Reactions were carried out in 40 mM HEPES-NaOH, pH 7.5, 80 mM NaCl, 0.5 mM MgCl₂ at 30°C. 250 nM protein or protein complex were pre-incubated with 50 nM DNA duplex for 5 min. 60 µl of the protein-DNA mixture were rapidly mixed with 60 µl of 4 mM ATP/MgCl₂, and the excited Alexa 488 fluorescence signal was recorded for 20 min using a 495 nm cutoff filter (KV 495, Schott). For each experiment, at least two individual traces were averaged, baseline-corrected by the fluorescence immediately after addition of ATP and normalized to the baseline-corrected maximum fluorescence. Data for ASCC3^HR,D611A-based unwinding had been reported previously²⁰ and are reproduced here to facilitate direct comparison. Data were plotted using GraphPad Prism 6.0 and fitted to a double exponential equation (fraction unwound = A_fast*(1 – exp(–k_fast * t)) + A_slow * (1 – exp(–k_slow * t)); A, total unwinding amplitude; k, unwinding rate constants [s^− 1]; t, time [s]).²⁵ Amplitude-weighted unwinding rate constants were calculated as k_uaw = (A_fast * k_fast² + A_slow * k_slow²) / (A_fast * k_fast + A_slow * k_slow).²⁵

ATPase assays

Thin layer chromatography (TLC)-based ATPase assays were performed using [α-³²P]ATP (Hartmann Analytic).^26,27 To quantify DNA-stimulated ATPase activity, 0.5 µM protein or protein complex were combined with 1 mM of a 43-nt ssDNA (5’-GGCCGCGAGCCGGAAATTTAATTATAAACCAGACCGTCTCCTC-3’). 0.5 µM protein or protein complex or equivalent protein-DNA mixtures were incubated with 1 mM [α-³²P]ATP in 50 mM HEPES-NaOH, pH 7.5, 80 mM NaCl, 5 mM MgCl₂, 2 mM DTT at 30°C for up to 60 min. 5 µl of sample were withdrawn at selected time points and reactions were quenched with 5 µl of 100 mM EDTA. 0.8 µl of the samples were spotted on a PEI-cellulose TLC plate and chromatographed with 1 M acetic acid, 0.5 M LiCl, 20% (v/v) ethanol. The corresponding ADP and ATP spots were visualized using a Storm 860 phosphorimager (GMI, USA) and quantified using ImageQuant software (version 5.2; Cytiva). Data were plotted and analyzed using Prism software (Graphpad, version 5), the ATPase activity was calculated as the number of hydrolyzed ATP molecules per protein molecule per minute, by fitting quantified data to the equation V = (A_fast * V_fast² + A_slow * V_slow²) / (A_fast * V_fast + A_slow * V_slow); A_fast and A_slow, amplitudes of ATP hydrolyzed in the rapid and slow phase, respectively; V_fast and V_slow, rates of the rapid and slow hydrolysis phases [min^− 1]; V, ATP hydrolyzed as a function of time [min^− 1].

Fluorescence microscopy

The sub-cellular localizations of the Flag/His-tagged versions of ASC1 were determined by immuno-fluorescence.⁴³ 293 cell lines expressing Flag-tagged ASC1 variants were grown on coverslips and fixed using 4% (v/v) paraformaldehyde for 20 min before permeabilization using 0.1% (v/v) Triton-X-100 in PBS for 20 min. Cells were blocked using PBS supplemented with 10% (v/v) fetal bovine serum (FBS) and 0.1% (v/v) Triton-X-100 for 1 h, then treated for 2 h with an FITC-conjugated α-Flag M2 antibody (Sigma Aldrich F4049; 1:200) diluted in PBS containing 10% FBS and 0.1% Triton-X-100. Cells were washed, and coverslips were mounted using mounting media containing DAPI. Cells were imaged using a Nikon Ti2 2-E inverted microscope.

Immuno-precipitation and western blotting

293 cells expressing N- or C-terminally Flag/His-tagged versions of full-length or truncated ASC1 or the Flag tag were lysed by sonication in IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.1% (v/v) Triton-X-100, 10% (v/v) glycerol and cOmplete™ protease inhibitors. Lysates were cleared of debris by centrifugation at 20,000 x g for 10 min, then the cleared lysates were incubated with α-Flag M2 magnetic beads (Sigma-Aldrich #M8823) for 2 h. The matrix was washed five times with IP buffer and complexes were eluted using 3xFlag peptide (Sigma Aldrich #SAE0194). Proteins were precipitated using 20% (w/v) trichloroacetic acid (TCA) and separated by SDS-PAGE. Western blotting was performed using antibodies against the Flag tag (Sigma-Aldrich F3165; 1:7500), ASCC1 (Proteintech #12301-1-AP; 1:500), ASCC2 (Proteintech #11529-1-AP; 1:1000) and ASCC3 (Proteintech #17627-1-AP; 1:1000).

For immuno-precipitation of HA-tagged ASC1 variants (ASC1^wt, ASC1^ΔZnF, ASC1^{L174A − L180A−I190A} or ASC1^{C171A − C184A}), the transfected 293T cells were resuspended in ice cold, high salt co-IP buffer (50 mM Tris-HCl, pH 7.9, 300 mM KCl, 10% [v/v] glycerol, 1% [w/v] Triton X-100, 1 mM DTT) supplemented with protease inhibitors. The cells were then lysed by sonication and allowed to rotate at -4°C to complete lysis. Lysates were cleared by centrifugation and diluted to 150 mM KCl using co-IP buffer without KCl. Anti-HA beads (Santa Cruz Biotechnology, sc-7392 AC) were then added to the samples, and after incubation at 4°C for 3.5 h, the beads were centrifuged and washed multiple times with 150 mM KCl co-IP buffer. Bound proteins were eluted with SDS-PAGE loading buffer and boiled before analysis via SDS-PAGE/western blot using antibodies against the HA-tag (Abcam EPR22819-101, 1:4000) and ASCC3 as described previously¹³.

MMS sensitivity assays

The wt and ASC1 KO PC-3 cells were plated on a 96-well plate with 3,500 cells per well. Cells were exposed to media containing variable concentrations of MMS for 24 h at 37°C. Then, cells were recovered with fresh culture medium for an additional 48 h at 37°C. Cell viability was measured by using the MTS assay (Promega).

Cryogenic electron microscopy

The ASCC3^HR-ASC1 complex was prepared freshly in buffer 20 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 1 mM DTT, and concentrated to 4.15 mg/ml using a 50k ultra centrifugal filter (Merck). The sample was supplemented with 0.01% (w/v) n-dodecyl β-maltoside promptly before vitrification. 3.8 µl of the sample were applied to glow-discharged holey carbon R1.2/1.3 copper grids (Quantifoil Microtools, Germany) and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher) equilibrated at 10°C and 100% humidity.

Data acquisition was conducted on a FEI Titan Krios G3i TEM operated at 300 kV equipped with a Falcon 3EC detector. Movies were taken for 40.57 s accumulating a total electron flux of ~ 40 el/Å² in counting mode at a calibrated pixel size of 0.832 Å/px distributed over 33 fractions.

CryoEM data analysis

All image analysis steps were done with cryoSPARC (version 3.2.2)⁴⁴. Movie alignment was done with patch motion correction generating Fourier-cropped micrographs (pixel size 1.664 Å/px), CTF estimation was conducted by Patch CTF. Class averages of manually selected particle images were used to generate an initial template for reference-based particle picking from 6,022 micrographs. 2,818,857 particle images were extracted with a box size of 160 px and Fourier-cropped to 80 px for initial analysis. Reference-free 2D classification was used to select 1,590,881 particle images for further analysis. Ab initio reconstruction using a small subset of particles was conducted to generate an initial 3D reference for consecutive iterations of 3D heterogeneous refinement. 597,971 particle images were re-extracted with a box of 160 px and subjected to non-uniform refinement followed by CTF refinement. Another heterogeneous refinement round was applied to select 473,863 particle images for re-extraction at full spatial resolution after local motion correction (box size 320 px, 0.832 Å/px). A final heterogeneous refinement run was conducted to select 244,064 particle images for non-uniform refinement and generate the final reconstruction at a global resolution of 3.4 Å, locally extending down to 2.5 Å.

Model building, refinement and analysis

AlphaFold-predicted models²⁴ of ASCC3^HR and of regions of ASC1 were manually placed in the cryoEM reconstruction and adjusted by rigid body fitting and segmental real-space refinement using Coot (version 0.8.9.1)⁴⁵. The model was refined by iterative rounds of real space refinement in PHENIX (version 1.17.1)⁴⁶ and manual adjustment in Coot. Manual adjustments also took advantage of locally refined, focused cryoEM reconstructions. The structural model was evaluated with Molprobity (version 4.5.1)⁴⁷. Interface areas were analyzed via the PISA server (version 1.52)⁴⁸. Structure figures were prepared using ChimeraX (version 1.4)⁴⁹ and PyMOL (version 1.8; Schrödinger, LLC).

DNA-protein cross-linking/mass spectrometry

UV cross-linking was employed to generated zero length cross-links between protein and bound ssDNA oligos (T₁₂, T₂₄, T₃₆, T₄₈). DNA oligos were 5’-end labeled using [γ-³²P]ATP and T4 polynucleotide kinase using a standard protocol. 10 µl reaction mixtures containing 100 nM (“1” in Fig. 5b) or 200 nM (“2” in Fig. 5b) protein or protein complex and 4.3 nM radio-labeled DNA probe were incubated in a 72-well microbatch plate (Greiner) in 50 mM HEPES-NaOH, pH 7.5, 80 mM NaCl, 5 mM MgCl₂, 2 mM DTT on ice for 5 min, then the samples were exposed to 254 nm UV irradiation for 10 min (Ultra-violet cross-linker, Amersham Life Science). Cross-linked samples were separated by SDS-PAGE and visualized by autoradiography using a Storm 860 phosphorimager.

For identifying cross-linked peptides and residues, 6.7 nM unlabeled T₄₈ ssDNA were cross-linked to 200 nM ASCC3^HR or ASCC3^HR-ASC1 in 48 x 10 µl reactions as above and ethanol precipitated. Subsequent analyses were conducted in duplicates. The pellets were dissolved in 50 µl 4 M urea and diluted to 1 M Urea with 50 mM Tris-HCl, pH 7.5. To digest the DNA, 1 µl Universal nuclease (Pierce) and 1 µl Nuclease P1 (New England Biolabs) were added to the samples, followed by incubation at 37°C for 3 h. Protein digestion was performed with 1 µg of trypsin (Promega) overnight at 37°C. The samples were acidified with formic acid (FA; final concentration 0.1% [v/v]), and acetonitrile (ACN) was added to 5% (v/v) final concentration. Non cross-linked nucleotides were depleted by C18 reversed-phase chromatography with Harvard Apparatus MicroSpin columns. Sample was eluted by stepwise application of 50% (v/v) and 80% (v/v) ACN. Cross-linked peptides were enriched over linear peptides by TiO2 self-packed tip columns with 5% (v/v) glycerol as a competitor as described previously⁵⁰. The samples were dried under vacuum and resuspended in 10 to 15 µl of 2% (v/v) ACN, 0.05% (v/v) trifluoroacetic acid. 7 or 8 µl (first or second analysis) were used for LC-MS analysis.

Chromatographic separation was achieved with Dionex Ultimate 3000 UHPLC (Thermo Fischer Scientific) coupled with a C18 column packed in-house (ReproSil-Pur 120 C18-AQ, 1.9/3 µm pore size, 75 µm inner diameter, 30 cm length, Dr. Maisch GmbH). The flow rate was set to 300 nl/min, and a 44 min linear gradient was formed with mobile phase A (0.1% [v/v] FA) and B (80% [v/v] ACN, 0.08% [v/v] FA) from 8% or 10% (first or second analysis) to 45% mobile phase B. Data acquisition of eluting peptides was performed with Orbitrap Exploris 480 (Thermo Fischer Scientific). The resolution for survey scans was set to 120,000, the maximum injection time to 60 ms, the automatic gain control target to 100% or 250% (first or second analysis) and the dynamic exclusion to 9 s. Analytes selected for fragmentation were isolated with a 1.6 m/z window and fragmented with a normalized collision energy of 28. MS/MS spectra were acquired with a resolution of 30,000, a maximum injection time of 120 ms and an automatic gain control target of 100%.

Cross-link data analysis of the resulting raw files was performed with the OpenNuXL node of OpenMS (version 3.0.0)⁵¹. Default general settings were used and the preset DNA-UV Extended was selected. The sequences of the proteins in the sample were provided as a database. The maximum length of DNA adducts was set to 3 and poly-T was used as sequence. The resulting .idxml files were used for annotation, and spectra were manually validated.

Data availability

The cryoEM reconstruction of the ASCC3^HR-ASC1 complex has been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb) under accession code EMD-15521 (https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-15521). Structure coordinates have been deposited in the RCSB Protein Data Bank (https://www.rcsb.org) with accession code 8ALZ (https://www.rcsb.org/structure/8ALZ).⁵² The DNA-protein CLMS data have been deposited in the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE⁵³ partner repository (https://www.ebi.ac.uk/pride/) under dataset identifier PXD036106 (https://www.ebi.ac.uk/pride/archive/projects/PXD036106). All other data are contained in the manuscript or the Supplementary Information. Source data are provided with this paper.

Acknowledgements

We thank Agnieszka Pietrzyk-Brzezinska for help in cloning of the ALKBH3 expression construct, Philipp Hackert for technical assistance, Ralf Pflanz and Monika Raabe for help in mass spectrometric analysis. We acknowledge the assistance of the core facility BioSupraMol supported by the Deutsche Forschungsgemeinschaft in electron microscopic analyses. This work was supported by grants from the Deutsche Forschungsgemeinschaft (INST 130/1064-1 FUGG to Freie Universität Berlin; GRK 2473 "Bioactive Peptides", project number 392923329, to M.C.W., BO3442/1-2 to M.T.B., SFB860 to K.E.B. and SFB860-A10 to H.U.), the American Cancer Society (RSG-18-156-01-DMC to N.M.), the National Institutes of Health of the U.S. (R01 CA193318 and P01 CA092584 to N.M.) and the Berlin University Alliance (501_BIS-CryoFac to M.C.W.).

Author contributions

J.J. cloned genes, purified proteins, assembled complexes for cryoEM and CLMS, conducted ATPase assays and generated stable cell lines. J.J., A.A. and N.H. performed in vitro interaction assays and unwinding experiments. T.H. acquired, processed and refined cryoEM data. J.J. and B.L. built and refined atomic models. K.E.B. generated stable cell lines for the inducible expression of Flag-tagged ASC1 variants and conducted cellular localization and pull-down analyses. L.P. generated stable cell lines for the expression of HA-tagged ASC1 variants and conducted pull-down analyses. N.T. created ASC1 KO cells and analyzed MMS sensitivity. A.C. and J.S. conducted DNA-protein CLMS analyses. All authors contributed to the analysis of the data and the interpretation of the results. J.J. and M.C.W. wrote the manuscript with contributions from the other authors. N.M., M.T.B., H.U. and M.C.W. supervised work in their respective groups and coordinated the collaboration.

Competing interests

The authors declare no competing interests.

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There is NO Competing Interest.

SupplementaryInformation.docx
Supplementary Tables and Figures
Sourcedata1gels.pdf
Supplementary Dataset 1
Sourcedata2unwindingATPaseUVCLMMSsurvival.xlsx
Supplementary Dataset 2
Sourcedata3DNAproteinCLMS.xlsx
Supplementary Dataset 3
ASCC3ASC1PDBvalidationreport.pdf
Supplementary Dataset 4
ASCC3ASC1.txt
Supplementary Dataset 5
ASCC3ASC1overall.mrc
Supplementary Dataset 6
ASCC3ASC1focussedonNC.mrc
Supplementary Dataset 7
ASCC3ASC1focussedonCC.mrc
Supplementary Dataset 8

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published 05 Apr, 2023

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

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Abstract

Figures

Introduction

Results

ASC1 directly associates with the helicase region of ASCC3

ASC1 activates ASCC3 helicase activity without influencing ASCC3 ATPase activity

DNA can be threaded through both ASCC3 helicase cassettes and along ASC1

ASC1 and ALKBH3 support ASCC core subunits in distinct cellular functions

Discussion

Methods

Molecular cloning

Generation of cell lines

CRISPR/Cas9-based genome editing

Recombinant protein production and purification

Analytical size exclusion chromatography

DNA unwinding assays

ATPase assays

Fluorescence microscopy

Immuno-precipitation and western blotting

MMS sensitivity assays

Cryogenic electron microscopy

CryoEM data analysis

Model building, refinement and analysis

DNA-protein cross-linking/mass spectrometry

Data availability

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

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