Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex

Wang, Joy Y.; Hoel, Christopher; Al-Shayeb, Basem; Banfield, Jillian F.; Brohawn, Stephen G.; Doudna, Jennifer A.

doi:10.1038/s41467-021-22900-y

Cited by 17 publications

(24 citation statements)

References 71 publications

(56 reference statements)

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“…They also provide a basis for future biotechnological applications. Our structures, together with structures of group II intron maturases and CRISPR-associated RT ( 32 , 38 , 39 ), also fill important gaps in our knowledge of very diverse bacterial RTs.…”

Section: Discussionmentioning

confidence: 80%

“…Instead, its position in space is occupied by the first three helices of the helical domain of Ll- AbiK. The recent structures of the Cas6-RT-Cas1 fusion protein from Thiomicrospira showed that this protein also lacks the thumb subdomain ( 32 ). Therefore, loss or replacement of the thumb subdomain can occur to accommodate various functions of the polymerase domain.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases

Figiel

Gapińska

Czarnocki-Cieciura

et al. 2022

Nucleic Acids Research

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Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases. They are unique in the way they combine the ability to synthesize DNA in a template-independent manner with protein priming. Here, we report crystal and cryo-electron microscopy structures of two Abi polymerases: AbiK and Abi-P2. Both proteins adopt a bilobal structure with an RT-like domain that comprises palm and fingers subdomains and a unique helical domain. AbiK and Abi-P2 adopt a hexameric and trimeric configuration, respectively, which is unprecedented for reverse transcriptases. Biochemical experiments showed that the formation of these oligomers is required for the DNA polymerization activity. The structure of the AbiK–DNA covalent adduct visualized interactions between the 3′ end of DNA and the active site and covalent attachment of the 5′ end of DNA to a tyrosine residue used for protein priming. Our data reveal a structural basis of the mechanism of highly unusual template-independent protein-priming polymerases.

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Section: Discussionmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases

Figiel

Gapińska

Czarnocki-Cieciura

et al. 2022

Nucleic Acids Research

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“…This crosstalk between the different components of RT–CRISPR systems has been validated by the cryo-EM structure of a Cas6RTCas1–Cas2 complex from Thiomicrospira (Wang et al . 2021 ), in which the linking RT helix regulates both RT and Cas1 activities.…”

Section: Introductionmentioning

confidence: 99%

Prokaryotic reverse transcriptases: from retroelements to specialized defense systems

González-Delgado

Mestre

Martı́nez-Abarca

et al. 2021

FEMS Microbiology Reviews

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Reverse transcriptases (RTs) catalyze the polymerization of DNA from an RNA template. These enzymes were first discovered in RNA tumor viruses in 1970, but it was not until 1989 that they were found in prokaryotes as a key component of retrons. Apart from RTs encoded by the “selfish” mobile retroelements known as group II introns, prokaryotic RTs are extraordinarily diverse, but their function has remained elusive. However, recent studies have revealed that different lineages of prokaryotic RTs, including retrons, those associated with CRISPR-Cas systems, Abi-like RTs, and other yet uncharacterized RTs, are key components of different lines of defense against phages and other mobile genetic elements. Prokaryotic RTs participate in various antiviral strategies, including abortive infection (Abi), in which the infected cell is induced to commit suicide to protect the host population, adaptive immunity, in which a memory of previous infection is used to build an efficient defense, and other as yet unidentified mechanisms. These prokaryotic enzymes are attracting considerable attention, both for use in cutting-edge technologies, such as genome editing, and as an emerging research topic. In this review, we discuss what is known about prokaryotic RTs, and the exciting evidence for their domestication from retroelements to create specialized defense systems.

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“…Interestingly, the RNA molecules could be ligated to either side of the first repeat of the array [ 57 ], which is consistent with the observed lack of orientation bias in the acquired spacers. On the other hand, the Cas1::RT-Cas2 complex from Thiomicrospira ligated RNA molecules to the CRISPR-containing DNA fragments in vitro but was unable to convert such intermediates to extended CRISPR arrays, suggesting that in vivo such conversion either requires additional factors, or that the cDNA synthesis must occur before the integration of prespacers into the CRISPR array, which should proceed in both orientations to explain the lack of spacer orientation bias [ 59 ].…”

Section: Spacer Acquisition In Type III Crispr-cas Systemsmentioning

confidence: 99%

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System

Kolesnik

Fedorova

Karneyeva

et al. 2021

Biochemistry Moscow

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The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit – the so-called CRISPR polymerase – which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.

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Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex

Cited by 17 publications

References 71 publications

Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases

Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases

Prokaryotic reverse transcriptases: from retroelements to specialized defense systems

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System

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