| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| anti-mAID | MBL International | Cat# M214-3, RRID:AB_2890014 |
| anti-GAPDH-HRP | Thermo Fisher Scientific | Cat# MA5-15738-HRP, RRID:AB_2537659 |
| Bacterial and virus strains | ||
| E. coli DH10 EMBacY | Geneva Biotech | N/A |
| E. coli TOP10 | Thermo Fisher | Cat# C404010 |
| Chemicals, peptides, and recombinant proteins | ||
| Auxin (3-Indoleacetic acid) | Sigma | Cat# I3750-100G-A |
| 4-thiouracil | Sigma | Cat# 440736–1G |
| 5-FOA | Zymo Research | Cat# F9001-5 |
| G418 | Sigma | Cat# A1720-5G |
| BioLock | IBA-Lifesciences | Cat# 2-0205-050 |
| Strep-Tactin resin | IBA-Lifesciences | Cat# 2-1201-025 |
| Desthiobiotin | IBA-Lifesciences | Cat# 2-1000-005 |
| Sulfo-SDA (sulfosuccinimidyl 4,4′-azipentanoate) | Thermo Fisher | Cat# 26173 |
| Instant Blue | Abcam | Cat# 119211 |
| Protease inhibitor tablets | Roche | Cat# 11836153001 |
| TRI reagent | Thermo Fisher | Cat# AM9738 |
| DnaseI (Rnase free) | New England Biolabs | Cat# M0303S |
| EZ-Link HPDP Biotin | Thermo Fisher | Cat# A35390 |
| Dynabeads MyOne Streptavidin C1 | Thermo Fisher | Cat# 65001 |
| FuGENE HD | Promega | Cat# E2311 |
| UltraPure Salmon sperm DNA solution | Invitrogen | Cat# 15632011 |
| LDS Sample Buffer | Pierce | Cat# 84788 |
| ECL Western Blotting Reagents | Cytiva | Cat# RPN2106 |
| GlycoBlue Coprecipitant | Thermo Fisher | Cat# AM9515 |
| Phenol:Chloroform:Iso-amyl alcohol (125:24:1) | Sigma | Cat# P1944-100ML |
| Recombinant protein: S. cerevisiae polymerase module | (Casañal et al., 2017) | N/A |
| Recombinant protein: S. cerevisiae polymerase module-Mpe1-SII | This study | N/A |
| Recombinant protein: S. cerevisiae polymerase module-Mpe1P215G-SII | This study | N/A |
| Recombinant protein: S. cerevisiae polymerase module-Mpe1W257A/Y260A-SII | This study | N/A |
| Recombinant protein: S. cerevisiae polymerase module-Mpe1-pcCYC1 | This study | N/A |
| Recombinant protein: S. cerevisiae polymerase module-Mpe1-Cft2(S)-pcCYC1 | This study | N/A |
| Recombinant protein: S. cerevisiae polymerase module-Mpe1-yPIM-pcCYC1 | This study | N/A |
| Recombinant protein: S. cerevisiae CPF | (Kumar et al., 2021), This study | N/A |
| Recombinant protein: S. cerevisiae nuclease-phosphatase modules (nuc-phos) | (Kumar et al., 2021) | N/A |
| Recombinant protein: S. cerevisiae Ysh1-Cft2-phosphatase module | This study | N/A |
| Recombinant protein: S. cerevisiae CPFΔMpe1 | This study | N/A |
| Recombinant protein: S. cerevisiae CPFP215G | This study | N/A |
| Recombinant protein: S. cerevisiae CPFW257A/Y260A | This study | N/A |
| Recombinant protein: S. cerevisiae CF IA | (Kumar et al., 2021) | N/A |
| Recombinant protein: S. cerevisiae CF IB | (Hill et al., 2019) | N/A |
| yPIM (peptide sequence): ASKHKMFPFNPAKIKKDDYGTVVDFTMFLPDDS | This study (GenScript) | N/A |
| Critical commercial assays | ||
| NEBNext Ultra II Directional RNA Library Prep Kit for Illumina | New England Biolabs | Cat# E7760S |
| NEBNext rRNA depletion kit | New England Biolabs | Cat# E6310S |
| Power SYBR Green PCR | Thermo Fisher | Cat# 4367659 |
| Phusion high-fidelity DNA Polymerase | New England Biolabs | Cat# M0530S |
| HiScribe T7 Quick High Yield RNA Synthesis kit | New England Biolabs | Cat# E2050S |
| Monarch RNA Cleanup kit | New England Biolabs | Cat# T2030S |
| RNA 6000 Nano Kit | Agilent | Cat# 5067-1511 |
| SuperScriptIII Reverse Transcriptase | Invitrogen | Cat# 18080-093 |
| Deposited data | ||
| RNA-seq of total and nascent RNA | This study | ArrayExpress: E-MTAB-10820 |
| RNA-seq after nuclear depletion of Ysh1 | (Baejen et al., 2017) | GEO: GSE79222 |
| Cross-linking mass spectrometry data | This study | ProteomeXchange: PXD027482 |
| Original images, chromatograms and qPCR data | This study | Mendeley Data: https://dx.doi.org/10.17632 |
| Polymerase module-Mpe1-RNA (EM map) | This study | EMDB: EMD-14710 |
| Polymerase module-Cft2(S) (EM map) | This study | EMDB: EMD-14711 |
| Polymerase module-Mpe1-yPIM-RNA (EM map) | This study | EMDB: EMD-14712 |
| Polymerase module-Mpe1-RNA (model) | This study | PDB: 7ZGP |
| Polymerase module-Cft2(S) (model) | This study | PDB: 7ZGQ |
| Polymerase module-Mpe1-yPIM-RNA (model) | This study | PDB: 7ZGR |
| Polymerase module | (Casañal et al., 2017) | EMDB: 3908 |
| Polymerase module | (Casañal et al., 2017) | PDB: 6eoj |
| mPSF-PIM | (Zhang et al., 2020) | PDB: 6urg |
| Pap1-Fip | (Meinke et al., 2008) | PDB: 3c66 |
| Experimental models: Cell lines | ||
| Sf9 | Oxford Expression Technologies Ltd. | Cat# 600100-Sf9 cells |
| Experimental models: Organisms/strains | ||
| S. cerevisiae: MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 can1-100 ura3-1∷ADH1-OsTIR1(pMK200, URA3) | (Nishimura and Kanemaki, 2014) | YMK728 (S2-31) |
| S. cerevisiae: YMK728 Mpe1-3miniAID-3FLAG | This study | JRY101 (S2-37) |
| S. cerevisiae: JRY101 pRS314 | This study | JRY200 (S3-64) |
| S. cerevisiae: JRY101 pRS314-MPE1 | This study | JRY208 (S4-24) |
| S. cerevisiae: JRY101 pRS314-mpe1(P215G) | This study | JRY210 (S4-26) |
| S. cerevisiae: YMK728 Mpe1-3mAID-3FLAG (OsTIR1-, URA3-) | This study | JRY114 (S2-52) |
| S. pombe: h+ | Juan Mata | JU60 (S3-30) |
| Oligonucleotides | ||
| Complete list of DNA oligonucleotide sequences | This study | Table S1 |
| precleaved CYC1 (pcCYC1): 5ʹ 6-FAM-UUUAUAGUUAUGUUAGUAUUAAGAA CGUUAUUUAUAUUUCAA 3′ |
(Casañal et al., 2017) | N/A |
|
CYC1: 5ʹ 6-FAM-UUUAUAGU UAUGUUAGUAUUAAGAACGUUAUUUAU AUUUCAAAUUUUUCUUUUUUU-A647 3′ |
(Hill et al., 2019) | CYC1a |
| Recombinant DNA | ||
| pRS314 | (Sikorski and Hieter, 1989) | P19-17 |
| pRS314-Mpe1 | This study | P34-48 |
| pRS314-Mpe1(P215G) | This study | P34-49 |
| pACEBac1-Mpe1(FDRP)-TEV-SII | This study | P24-58 |
| (modified) pBig1A | (Hill et al., 2019; Weissmann et al., 2016) | P24-63 |
| (modified) pBig1B | (Hill et al., 2019; Weissmann et al., 2016) | P24-64 |
| (modified) pBig2AB | (Hill et al., 2019; Weissmann et al., 2016) | P25-3 |
| pACEBac1-Cft1 | (Casañal et al., 2017) | P14-39 |
| pACEBac1-Pfs2-SII | (Casañal et al., 2017) | P14-40 |
| pACEBac1-Yth1 | (Casañal et al., 2017) | P14-42 |
| pACEBac1-Mpe1(P215G)-TEV-SII | This study | P31-24 |
| pACEBac1-Mpe1(W257A, Y260A)-TEV-SII | This study | P31-25 |
| pACEBac1-Cft2-SII | (Hill et al., 2019) | P25-7 |
| pACEBac1-Cft2(F537A, Y549A, F558A)-TEV-SII | This study | P34-45 |
| pACEBac1-Mpe1 (ZnK-PSR)-TEV-SII | This study | P27-60 |
| pACEBac1-Mpe1ΔPSR-TEV-SII | This study | P34-47 |
| pACEBac1-Mpe1ΔZnK-TEV-SII | This study | P34-46 |
| pIDC-Fip1 | (Casañal et al., 2017) | P14-44 |
| pIDC-Pap1 | (Casañal et al., 2017) | P14-45 |
| pIDS-Mpe1-SII | (Hill et al., 2019) | P14-56 |
| pIDS-Ysh1 | (Hill et al., 2019) | P14-59 |
| pIDS-Cft2 | (Hill et al., 2019) | P14-57 |
| pBig1A-Cft1-Mpe1-SII | This study | P25-38 |
| pBig1A-Pap1-Mpe1-SII | This study | P25-39 |
| pBig1A-Pfs2-Mpe1-SII | This study | P25-40 |
| pBig1A-Fip1-Mpe1-SII | This study | P25-41 |
| pBig1A-Yth1-Mpe1-SII | This study | P25-42 |
| pBig1A-Construct A (Cft1-Pap1-Pfs2-Fip1-Yth1) | (Hill et al., 2019) | P20-1 |
| pBig1B-Mpe1-SII | This study | P25-41 |
| pBig1A-Construct B (Cft1-Pap1-Pfs2-SII-Fip1-Yth1) | (Hill et al., 2019) | P20-3 |
| pBig2AB-Construct A + Mpe1-SII | This study | P26-20 |
| pBig1B-Construct AX (Ysh1-Cft2) | This study | P20-54 |
| pBig2AB-Ssu72-Pti1-Glc7-Ref2-SII-Swd2 | (Kumar et al., 2021) | P27-37 |
| pET28a +(modified) 6H-3C-Cft2(short) | Chris Hill | P19-8 |
| Software and algorithms | ||
| Integrated Genome Viewer (v. 2.4.11) | (Robinson et al., 2011) | https://software.broadinstitute.org/software/igv/ |
| RUV-seq (v. 1.20.0) | (Risso et al., 2014) | https://bioconductor.org/packages/release/bioc/html/RUVSeq.html |
| Rsubread (v. 2.0.1) | (Liao et al., 2014) | https://bioconductor.org/packages/release/bioc/html/Rsubread.html |
| STAR (v. 2.6.0a) | (Dobin et al., 2013) | https://github.com/alexdobin/STAR |
| TrimGalore (v. 0.4.5) | https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ | https://github.com/FelixKrueger/TrimGalore |
| SAMtools | (Li et al., 2009) | http://samtools.sourceforge.net/ |
| Deeptools (v. 3.1.3) | (Ramirez et al., 2016) | https://deeptools.readthedocs.io/en/develop/ |
| R (v. 3.6.0) | (R Core Team, 2019) | https://www.r-project.org |
| DESeq2 (v. 1.26.0) | (Love et al., 2014) | https://bioconductor.org/packages/release/bioc/html/DESeq2.html |
| SeqPlots | (Stempor and Ahringer, 2016) | https://github.com/Przemol/seqplots |
| Prism 8 (v. 8.1.2) | N/A | https://www.graphpad.com |
| cryoEF | (Naydenova and Russo, 2017) | https://www.mrc-lmb.cam.ac.uk/crusso/cryoEF/ |
| ProtParam | (Gasteiger, 2005) | https://web.expasy.org/protparam/ |
| Relion 3.1 | (Zivanov et al., 2018) | https://github.com/3dem/relion |
| DynamX | Waters | N/A |
| Coot (v. 0.9.5.1-pre) | (Emsley and Cowtan, 2004; Emsley et al., 2010) | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot |
| ChimeraX (v. 1.2) | (Goddard et al., 2018) | https://www.cgl.ucsf.edu/chimerax/ |
| PDBePISA | (Krissinel and Henrick, 2007) | https://www.ebi.ac.uk/pdbe/pisa/ |
| ClustalW | (Goujon et al., 2010; Sievers et al., 2011) | https://www.ebi.ac.uk/Tools/msa/clustalo/ |
| ImageJ (v. 1.52a) | (Schneider et al., 2012) | https://imagej.net/software/imagej/ |
| Jalview (v 1.0) | (Clamp et al., 2004) | http://www.compbio.dundee.ac.uk/ftp/embnet.news/vol5_4/embnet/body_jalview.html |
| Other | ||
| Insect-XPRESS™ Protein-free Insect cell medium | Lonza | Cat# BELN12-730Q |
| MonoQ 5/50 GL | Cytiva | Cat# 17516601 |
| HiTrap Heparin 1ml | Cytiva | Cat# 17040601 |
| HiLoad 16/600 Superdex 200 pg | Cytiva | Cat# 28989335 |
| UltrAuFoil R 1.2/1.3 on Au 300 mesh grids | Quantifoil | Cat# N1-A14nAu30-50 |
| Superose 6 Increase 3.2/300 | Cytiva | Cat# 29091598 |
| Superdex 30 Increase 3.2/300 | Cytiva | Cat# 29219758 |
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