CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

Domgaard, Hannah; Cahoon, Christian; Armbrust, Matthew J.; Redman, Olivine; Jolley, Alivia; Thomas, Aaron; Jackson, Ryan N.

doi:10.1093/nar/gkad546

Title: CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

Journal Article · 02 July 2023 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkad546 · OSTI ID:1987970

Domgaard, Hannah;

; Armbrust, Matthew J.;

; Jolley, Alivia; Thomas, Aaron;

Abstract CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. Here, we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP-dependent 5′-3′ DNA translocase that unwinds double-stranded (ds)DNA and RNA/DNA hybrids. The crystal structure of CasDinG reveals a superfamily 2 helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these domains, we identified the preferred PAM sequence for the type IV-A system (5′-GNAWN-3′ on the 5′-side of the target) with a plasmid library and performed plasmid clearance assays with domain deletion mutants. Plasmid clearance assays demonstrated that all three domains are essential for type IV-A immunity. Protein expression and biochemical assays suggested the vFeS domain is needed for protein stability and the arch for helicase activity. However, deletion of the N-terminal domain did not impair ATPase, ssDNA binding, or helicase activities, indicating a role distinct from canonical helicase activities that structure prediction tools suggest involves interaction with dsDNA. This work demonstrates CasDinG helicase activity is essential for type IV-A CRISPR immunity as well as the yet undetermined activity of the CasDinG N-terminal domain.

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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

Sponsoring Organization:: National Institute of General Medical Sciences (NIGMS); USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1987970

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 15 Vol. 51; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United Kingdom

Language:: English

References (64)

Crystal structure analysis of a hypothetical protein (MJ0366) from Methanocaldococcus jannaschii revealed a novel topological arrangement of the knot fold Thiruselvam, Viswanathan; Kumarevel, Thirumananseri; Karthe, Ponnuraj Biochemical and Biophysical Research Communications, Vol. 482, Issue 2 https://doi.org/10.1016/j.bbrc.2016.11.052	journal	January 2017
Structure of the DNA Repair Helicase XPD Liu, Huanting; Rudolf, Jana; Johnson, Kenneth A. Cell, Vol. 133, Issue 5 https://doi.org/10.1016/j.cell.2008.04.029	journal	May 2008
XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations Fan, Li; Fuss, Jill O.; Cheng, Quen J. Cell, Vol. 133, Issue 5 https://doi.org/10.1016/j.cell.2008.04.030	journal	May 2008
Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System Redding, Sy; Sternberg, Samuel H.; Marshall, Myles Cell, Vol. 163, Issue 4 https://doi.org/10.1016/j.cell.2015.10.003	journal	November 2015
Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering Wright, Addison V.; Nuñez, James K.; Doudna, Jennifer A. Cell, Vol. 164, Issue 1-2 https://doi.org/10.1016/j.cell.2015.12.035	journal	January 2016
The Biology of CRISPR-Cas: Backward and Forward Hille, Frank; Richter, Hagen; Wong, Shi Pey Cell, Vol. 172, Issue 6 https://doi.org/10.1016/j.cell.2017.11.032	journal	March 2018
Inference of Macromolecular Assemblies from Crystalline State Krissinel, Evgeny; Henrick, Kim Journal of Molecular Biology, Vol. 372, Issue 3 https://doi.org/10.1016/j.jmb.2007.05.022	journal	September 2007
Conformational regulation of CRISPR-associated nucleases Jackson, Ryan N.; van Erp, Paul BG; Sternberg, Samuel H. Current Opinion in Microbiology, Vol. 37 https://doi.org/10.1016/j.mib.2017.05.010	journal	June 2017
The DNA Repair Helicases XPD and FancJ Have Essential Iron-Sulfur Domains Rudolf, Jana; Makrantoni, Vasso; Ingledew, W. John Molecular Cell, Vol. 23, Issue 6 https://doi.org/10.1016/j.molcel.2006.07.019	journal	September 2006
CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3 Westra, Edze R.; van Erp, Paul B. G.; Künne, Tim Molecular Cell, Vol. 46, Issue 5 https://doi.org/10.1016/j.molcel.2012.03.018	journal	June 2012
Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems Leenay, Ryan T.; Maksimchuk, Kenneth R.; Slotkowski, Rebecca A. Molecular Cell, Vol. 62, Issue 1 https://doi.org/10.1016/j.molcel.2016.02.031	journal	April 2016
SF1 and SF2 helicases: family matters Fairman-Williams, Margaret E.; Guenther, Ulf-Peter; Jankowsky, Eckhard Current Opinion in Structural Biology, Vol. 20, Issue 3 https://doi.org/10.1016/j.sbi.2010.03.011	journal	June 2010
Molecular Mechanism Underlying Inhibition of Intrinsic ATPase Activity in a Ski2-like RNA Helicase Absmeier, Eva; Santos, Karine F.; Wahl, Markus C. Structure, Vol. 28, Issue 2 https://doi.org/10.1016/j.str.2019.11.014	journal	February 2020
Solution Structure of Dimeric Mnt Repressor (1-76) Burgering, Maurits J. M.; Boelens, Rolf; Gilbert, Dara E. Biochemistry, Vol. 33, Issue 50 https://doi.org/10.1021/bi00254a012	journal	December 1994
DNA recognition by β-sheets in the Arc represser–operator crystal structure Raumann, Brigitte E.; Rould, Mark A.; Pabo, Carl O. Nature, Vol. 367, Issue 6465 https://doi.org/10.1038/367754a0	journal	February 1994
Structural insights into DndE from Escherichia coli B7A involved in DNA phosphorothioation modification Hu, Wei; Wang, Chengkun; Liang, Jingdan Cell Research, Vol. 22, Issue 7 https://doi.org/10.1038/cr.2012.66	journal	April 2012
Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system: Cas3 nuclease/helicase Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe The EMBO Journal, Vol. 30, Issue 7 https://doi.org/10.1038/emboj.2011.41	journal	February 2011
Self versus non-self discrimination during CRISPR RNA-directed immunity Marraffini, Luciano A.; Sontheimer, Erik J. Nature, Vol. 463, Issue 7280 https://doi.org/10.1038/nature08703	journal	January 2010
The cryo-electron microscopy structure of human transcription factor IIH Greber, Basil J.; Nguyen, Thi Hoang Duong; Fang, Jie Nature, Vol. 549, Issue 7672 https://doi.org/10.1038/nature23903	journal	September 2017
In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair Peissert, Stefan; Sauer, Florian; Grabarczyk, Daniel B. Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-15241-9	journal	April 2020
Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum Özcan, Ahsen; Pausch, Patrick; Linden, Andreas Nature Microbiology, Vol. 4, Issue 1 https://doi.org/10.1038/s41564-018-0274-8	journal	November 2018
Characterization of the self-targeting Type IV CRISPR interference system in Pseudomonas oleovorans Guo, Xiaohan; Sanchez-Londono, Mariana; Gomes-Filho, José Vicente Nature Microbiology, Vol. 7, Issue 11 https://doi.org/10.1038/s41564-022-01229-2	journal	September 2022
Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants Makarova, Kira S.; Wolf, Yuri I.; Iranzo, Jaime Nature Reviews Microbiology, Vol. 18, Issue 2 https://doi.org/10.1038/s41579-019-0299-x	journal	December 2019
Fast and accurate protein structure search with Foldseek van Kempen, Michel; Kim, Stephanie S.; Tumescheit, Charlotte Nature Biotechnology https://doi.org/10.1038/s41587-023-01773-0	journal	May 2023
ColabFold: making protein folding accessible to all Mirdita, Milot; Schütze, Konstantin; Moriwaki, Yoshitaka Nature Methods, Vol. 19, Issue 6 https://doi.org/10.1038/s41592-022-01488-1	journal	May 2022
DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks Gan, Rui; Wu, Xiaolin; He, Wei Scientific Reports, Vol. 4, Issue 1 https://doi.org/10.1038/srep06642	journal	October 2014
Staphylococcus aureus DinG, a helicase that has evolved into a nuclease McRobbie, Anne-Marie; Meyer, Bjoern; Rouillon, Christophe Biochemical Journal, Vol. 442, Issue 1 https://doi.org/10.1042/BJ20111903	journal	January 2012
Structure, function and evolution of the XPD family of iron–sulfur-containing 5′→3′ DNA helicases White, Malcolm F. Biochemical Society Transactions, Vol. 37, Issue 3 https://doi.org/10.1042/BST0370547	journal	May 2009
Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Tabtiang, R. K.; Cezairliyan, B. O.; Grant, R. A. Proceedings of the National Academy of Sciences, Vol. 102, Issue 7 https://doi.org/10.1073/pnas.0409562102	journal	February 2005
Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold Lu, J.; den Dulk-Ras, A.; Hooykaas, P. J. J. Proceedings of the National Academy of Sciences, Vol. 106, Issue 24 https://doi.org/10.1073/pnas.0812199106	journal	May 2009
Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene Taylor, E. M.; Broughton, B. C.; Botta, E. Proceedings of the National Academy of Sciences, Vol. 94, Issue 16 https://doi.org/10.1073/pnas.94.16.8658	journal	August 1997
In Vitro Reconstitution of an Escherichia coli RNA-guided Immune System Reveals Unidirectional, ATP-dependent Degradation of DNA Target Mulepati, Sabin; Bailey, Scott Journal of Biological Chemistry, Vol. 288, Issue 31 https://doi.org/10.1074/jbc.M113.472233	journal	June 2013
Characterization of the DNA Damage-inducible Helicase DinG from Escherichia coli Voloshin, Oleg N.; Vanevski, Filip; Khil, Pavel P. Journal of Biological Chemistry, Vol. 278, Issue 30 https://doi.org/10.1074/jbc.M301188200	journal	July 2003
ATP Binding Modulates the Nucleic Acid Affinity of Hepatitis C Virus Helicase Levin, Mikhail K.; Gurjar, Madhura M.; Patel, Smita S. Journal of Biological Chemistry, Vol. 278, Issue 26 https://doi.org/10.1074/jbc.M301283200	journal	June 2003
The DinG Protein from Escherichia coli Is a Structure-specific Helicase Voloshin, Oleg N.; Camerini-Otero, R. Daniel Journal of Biological Chemistry, Vol. 282, Issue 25 https://doi.org/10.1074/jbc.M700376200	journal	June 2007
The Iron-containing Domain Is Essential in Rad3 Helicases for Coupling of ATP Hydrolysis to DNA Translocation and for Targeting the Helicase to the Single-stranded DNA-Double-stranded DNA Junction Pugh, Robert A.; Honda, Masayoshi; Leesley, Haley Journal of Biological Chemistry, Vol. 283, Issue 3 https://doi.org/10.1074/jbc.M707064200	journal	January 2008
A Type IV-A CRISPR-Cas System in Pseudomonas aeruginosa Mediates RNA-Guided Plasmid Interference In Vivo Crowley, Valerie M.; Catching, Adam; Taylor, Hannah N. The CRISPR Journal, Vol. 2, Issue 6 https://doi.org/10.1089/crispr.2019.0048	journal	December 2019
Evolution of Type IV CRISPR-Cas Systems: Insights from CRISPR Loci in Integrative Conjugative Elements of Acidithiobacillia Moya-Beltrán, Ana; Makarova, Kira S.; Acuña, Lillian G. The CRISPR Journal, Vol. 4, Issue 5 https://doi.org/10.1089/crispr.2021.0051	journal	October 2021
Escherichia colidinG gene encodes a putative DNA helicase related to a group of eukaryotic helicases including Rad3 protein Koonin, Eugene V. Nucleic Acids Research, Vol. 21, Issue 6 https://doi.org/10.1093/nar/21.6.1497	journal	January 1993
Search and sequence analysis tools services from EMBL-EBI in 2022 Madeira, Fábio; Pearce, Matt; Tivey, Adrian R. N. Nucleic Acids Research, Vol. 50, Issue W1 https://doi.org/10.1093/nar/gkac240	journal	April 2022
Dali server: structural unification of protein families Holm, Liisa Nucleic Acids Research, Vol. 50, Issue W1 https://doi.org/10.1093/nar/gkac387	journal	May 2022
ExPASy: the proteomics server for in-depth protein knowledge and analysis Gasteiger, E. Nucleic Acids Research, Vol. 31, Issue 13 https://doi.org/10.1093/nar/gkg563	journal	July 2003
DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data Whitmore, L.; Wallace, B. A. Nucleic Acids Research, Vol. 32, Issue S2, p. W668-W673 https://doi.org/10.1093/nar/gkh371	journal	July 2004
Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family Nongkhlaw, Macmillan; Dutta, Popy; Hockensmith, Joel W. Nucleic Acids Research, Vol. 37, Issue 10 https://doi.org/10.1093/nar/gkp178	journal	March 2009
A new bioinformatics analysis tools framework at EMBL-EBI Goujon, M.; McWilliam, H.; Li, W. Nucleic Acids Research, Vol. 38, Issue Web Server https://doi.org/10.1093/nar/gkq313	journal	May 2010
Type IV CRISPR–Cas systems are highly diverse and involved in competition between plasmids Pinilla-Redondo, Rafael; Mayo-Muñoz, David; Russel, Jakob Nucleic Acids Research, Vol. 48, Issue 4 https://doi.org/10.1093/nar/gkz1197	journal	December 2019
Structural insights into the modulatory role of the accessory protein WYL1 in the Type VI-D CRISPR-Cas system Zhang, Heng; Dong, Cheng; Li, Li Nucleic Acids Research, Vol. 47, Issue 10 https://doi.org/10.1093/nar/gkz269	journal	April 2019
Short motif sequences determine the targets of the prokaryotic CRISPR defence system Mojica, F. J. M.; Díez-Villaseñor, C.; García-Martínez, J. Microbiology, Vol. 155, Issue 3, p. 733-740 https://doi.org/10.1099/mic.0.023960-0	journal	March 2009
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions Krissinel, E.; Henrick, K. Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12 https://doi.org/10.1107/S0907444904026460	journal	November 2004
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8 https://doi.org/10.1107/S0907444906019949	journal	July 2006
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Phaser.MRage : automated molecular replacement Bunkóczi, Gábor; Echols, Nathaniel; McCoy, Airlie J. Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 11 https://doi.org/10.1107/S0907444913022750	journal	October 2013
SOLVE and RESOLVE: automated structure solution, density modification and model building Terwilliger, Thomas Journal of Synchrotron Radiation, Vol. 11, Issue 1 https://doi.org/10.1107/S0909049503023938	journal	November 2003
Isolation of DNA damage-inducible promoters in Escherichia coli: regulation of polB (dinA), dinG, and dinH by LexA repressor Lewis, L. K.; Jenkins, M. E.; Mount, D. W. Journal of Bacteriology, Vol. 174, Issue 10 https://doi.org/10.1128/jb.174.10.3377-3385.1992	journal	May 1992
SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System Wang, Shiwei; Wan, Mengping; Huang, Ruolin mBio, Vol. 12, Issue 2 https://doi.org/10.1128/mBio.00613-21	journal	April 2021
Structural and Functional Analysis of DndE Involved in DNA Phosphorothioation in the Haloalkaliphilic Archaea Natronorubrum bangense JCM10635 He, Wei; Gao, Haiyan; Wu, Dan mBio, Vol. 13, Issue 3 https://doi.org/10.1128/mbio.00716-22	journal	June 2022
Translocation and Unwinding Mechanisms of RNA and DNA Helicases Pyle, Anna Marie Annual Review of Biophysics, Vol. 37, Issue 1 https://doi.org/10.1146/annurev.biophys.37.032807.125908	journal	June 2008
Interactive metagenomic visualization in a Web browser Ondov, Brian D.; Bergman, Nicholas H.; Phillippy, Adam M. BMC Bioinformatics, Vol. 12, Issue 1 https://doi.org/10.1186/1471-2105-12-385	journal	September 2011
Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition Westra, Edze R.; Semenova, Ekaterina; Datsenko, Kirill A. PLoS Genetics, Vol. 9, Issue 9 https://doi.org/10.1371/journal.pgen.1003742	journal	September 2013
Identification of a Type IV-A CRISPR-Cas System Located Exclusively on IncHI1B/IncFIB Plasmids in Enterobacteriaceae Newire, Enas; Aydin, Alp; Juma, Samina Frontiers in Microbiology, Vol. 11 https://doi.org/10.3389/fmicb.2020.01937	journal	August 2020
Positioning Diverse Type IV Structures and Functions Within Class 1 CRISPR-Cas Systems Taylor, Hannah N.; Laderman, Eric; Armbrust, Matt Frontiers in Microbiology, Vol. 12 https://doi.org/10.3389/fmicb.2021.671522	journal	May 2021
DNA translocation mechanism of an XPD family helicase Cheng, Kaiying; Wigley, Dale B. eLife, Vol. 7 https://doi.org/10.7554/eLife.42400	journal	December 2018
Uncovering an allosteric mode of action for a selective inhibitor of human Bloom syndrome protein Chen, Xiangrong; Ali, Yusuf I.; Fisher, Charlotte EL eLife, Vol. 10 https://doi.org/10.7554/eLife.65339	journal	March 2021

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Title: CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

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