title: Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3.
abstract: Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.
authors.affiliation: Interdisciplinary Graduate Program in Molecular Medicine, Chonnam National University, Gwangju 501-746, Korea;
authors.forename: Bei
authors.initials: B
authors.lastname: Gong
authors.affiliation: Department of Chemistry, Chonnam National University, Gwangju 500-757, Korea;
authors.forename: Minsang
authors.initials: M
authors.lastname: Shin
authors.affiliation: Interdisciplinary Graduate Program in Molecular Medicine, Chonnam National University, Gwangju 501-746, Korea;
authors.forename: Jiali
authors.initials: J
authors.lastname: Sun
authors.affiliation: Interdisciplinary Graduate Program in Molecular Medicine, Chonnam National University, Gwangju 501-746, Korea; Department of Chemistry, Chonnam National University, Gwangju 500-757, Korea;
authors.forename: Che-Hun
authors.initials: CH
authors.lastname: Jung
authors.affiliation: School of Life Sciences, University of Nottingham Medical School, Nottingham NG72UH, United Kingdom; and.
authors.forename: Edward L
authors.initials: EL
authors.lastname: Bolt
authors.affiliation: Laboratory of Microbiology, Wageningen University, 6703 HB, Wageningen, The Netherlands.
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
authors.affiliation: Interdisciplinary Graduate Program in Molecular Medicine, Chonnam National University, Gwangju 501-746, Korea; Department of Chemistry, Chonnam National University, Gwangju 500-757, Korea; jsunkim@chonnam.ac.kr.
authors.forename: Jeong-Sun
authors.initials: JS
authors.lastname: Kim
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000502:physiology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D020071:Interspersed Repetitive Sequences; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D008345:Manganese; D000255:Adenosine Triphosphate; D004247:DNA; D003851:Deoxyribonucleases; D004265:DNA Helicases; D008274:Magnesium
keywords: CRISPR; Cas proteins; Cas3; Cascade; bacterial immunity