Mechanism of Foreign DNA Selection in a Bacterial Adaptive Immune System

Sashital, Dipali G.; Wiedenheft, Blake; Doudna, Jennifer A.

doi:10.1016/j.molcel.2012.03.020

Cited by 231 publications

(260 citation statements)

References 48 publications

Supporting

Mentioning

244

Contrasting

Order By: Relevance

“…4). After recognition of an appropriate PAM motif by Cse1 (30), the crRNA-Cascade complex unwinds invading dsDNA in a step-wise manner (44), eventually generating an R-loop conformation with a displaced noncomplementary DNA strand and a complementary DNA strand that base pairs with the crRNA guide (28) (Fig. 4A).…”

Section: Discussionmentioning

confidence: 99%

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Gong

Shin

Sun

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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 HDnucleolytic 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.M any bacteria and archaea can eliminate invading phages and plasmids by activating a defense system that is based on processing of clustered regularly interspaced short palindromic repeats (CRISPRs) by CRISPR-associated (Cas) proteins and various additional proteins. These CRISPR-Cas systems are diverse in structure and function, but common principles of action allow their classification into three major types (I, II, and III) with some further divisions into subtypes (e.g., type IA-F) (1-6).Common CRISPR-mediated immunity processes are usually defined into three stages: (i) acquisition of a short DNA segment (protospacer) from an invading virus or plasmid, and its insertion at the leader-proximal end of a CRISPR locus (7, 8), (ii) generation of small mature CRISPR RNAs (crRNAs) from a longer transcript of a CRISPR locus (9-11), and (iii) interference of foreign nucleic acid invaders by a crRNA-containing ribonucleoprotein effector complex (12-18).Different interference effector complexes characterize the three major CRISPR types. Cascade (Cas complex for antiviral defense) complexes are synonymous with interference in type I CRISPRs, comprising multiple proteins in a helical structure around crRNA that targets invader DNA (13,19, 20). Type II CRISPR systems are characterized by Cas9, a multidomain protein with a bilobed architecture harboring two nuclease sites that together catalyze cleavage of invading DNA (12, 21-24). The Csm system in CRISPR type IIIA and a related Cmr ...

show abstract

Section: Discussionmentioning

confidence: 99%

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Gong

Shin

Sun

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Interference module proteins are diverse, and this diversity forms a basis of classification of CRISPR-Cas systems into two classes and several types (4). In DNA-targeting type I and type II CRISPR-Cas systems, target recognition requires, in addition to crRNA spacer-target protospacer complementarity, a protospacer adjacent motif (PAM) (8,9) recognized by effector proteins (10)(11)(12). Upon target DNA recognition, a stable R-loop containing locally melted protospacer DNA and an RNA-DNA heteroduplex is formed (13,14).…”

mentioning

confidence: 99%

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

Semenova

Savitskaya

Musharova

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

111

View full text Add to dashboard Cite

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated (Cas) immunity relies on adaptive acquisition of spacers-short fragments of foreign DNA. For the type I-E CRISPR-Cas system from Escherichia coli, efficient "primed" adaptation requires Cas effector proteins and a CRISPR RNA (crRNA) whose spacer partially matches a segment (protospacer) in target DNA. Primed adaptation leads to selective acquisition of additional spacers from DNA molecules recognized by the effector-crRNA complex. When the crRNA spacer fully matches a protospacer, CRISPR interference-that is, target destruction without acquisition of additional spacers-is observed. We show here that when the rate of degradation of DNA with fully and partially matching crRNA targets is made equal, fully matching protospacers stimulate primed adaptation much more efficiently than partially matching ones. The result indicates that different functional outcomes of CRISPR-Cas response to two kinds of protospacers are not caused by different structures formed by the effector-crRNA complex but are due to the more rapid destruction of targets with fully matching protospacers.CRISPR-Cas | CRISPR interference | primed CRISPR adaptation

show abstract

“…The CRISPR array is the most susceptible to autoimmune events as it contains spacers used for target recognition. In most cases, the CRISPR-Cas system deals with this problem by utilizing protospacer adjacent motifs (PAMs) (Sashital et al, 2012),which are present in the target DNA but not the CRISPR array. DNA cleavage occurs only if the correct PAM sequence is present (Jinek et al, 2012; Semenova et al, 2011;Westra et al, 2013) is PAM-independent and is based on the proximity of a repeat sequence to a spacer sequence in the CRISPR locus, which hinders its cleavage (Marraffini and Sontheimer, 2010).…”

Section: Structure Of the Crispr-cas Systemmentioning

confidence: 99%

“…Further hybridization to the protospacer region results in formation of an R-loop structure, in which the crRNA is paired with one of the DNA strands and the displaced DNA strand remains single-stranded (Sashital et al, 2012;Sorek et al, 2013). This structure triggers a conformational change in the crRNP complex, thereby initiating type-dependent nuclease activity Spilman et al, 2013;Wiedenheft et al, 2011).…”

Section: Target Recognition and Degradationmentioning

confidence: 99%

“…The Cse1 protein located at the 5' crRNA end of the complex is responsible for the non-specific interaction with DNA during initial target scanning . This protein is also responsible for interaction with the PAM motif located at the 3' end of the proto spacer , and this interaction is thought to trigger hybridization of the crRNA and target DNA by destabilization of the DNA duplex (Sashital et al, 2012). In type I-E systems, the seed region, which requires perfect hybridization for target degradation, is located at the 5' end of the spacer (positions 1-5, Burmistrz M. and Pyrć K. 3 198 7, and 8) Semenova et al, 2011).…”

mentioning

confidence: 99%

See 1 more Smart Citation

CRISPR-Cas Systems in Prokaryotes

Burmistrz

Pyrć

2015

Pol J Microbiol

View full text Add to dashboard Cite

A b s t r a c tProkaryotic organisms possess numerous strategies that enable survival in hostile conditions. Among others, these conditions include the invasion of foreign nucleic acids such as bacteriophages and plasmids. The clustered regularly interspaced palindromic repeats-CRISPRassociated proteins (CRISPR-Cas) system provides the majority of bacteria and archaea with adaptive and hereditary immunity against this threat. This mechanism of immunity is based on short fragments of foreign DNA incorporated within the hosts genome. After transcription, these fragments guide protein complexes that target foreign nucleic acids and promote their degradation. The aim of this review is to summarize the current status of CRISPR-Cas research, including the mechanisms of action, the classification of different types and subtypes of these systems, and the development of new CRISPR-Cas-based molecular biology tools. K e y w o r d s: CRISPR-Cas, prokaryotesBurmistrz M. and Pyrć K. 3 194 spacer sequences can be of either foreign or self-origin (Stern et al., 2010;Vercoe et al., 2013). The proteomic component is responsible for the incorporation of new template sequences, processing them into a form that enables base pairing with target nucleic acids, as well as for scanning and cleavage of target DNA or RNA. Of note, not all CRISPR-Cas systems discovered to date are active (Haft et al., 2005;van der Ploeg, 2009). Structure of the CRISPR-Cas systemThe genomic component of the CRISPR-Cas system is formed by a series of variable spacers, which in some cases share sequence similarity with viruses, plasmids, or bacteria. These regions are interspaced with repeat sequences that are identical or almost identical within a single CRISPR cassette. The length of the repeat sequences varies between 25 and 40 nt, whereas the length of the spacer sequences varies between 21 and 72 nt. As mentioned above, some spacers show high homology with foreign nucleic acids, but the origin of a significant percentage of spacers remains unknown. The sequence homology between the spacer and the target is the major determinant of nucleic acid degradation of the target. Some bacterial species contain more than one CRISPR locus within their genome (Louwen et al., 2014). Depending on the specific bacterial species or strain, a CRISPR locus may contain from a few to several hundred repeat spacer units; however, most commonly, a single CRISPR locus contains approximately 50 units. The CRISPR repeat sequences play an important role during both the acquisition of new spacers and the transcription and maturation of CRISPR RNA (crRNA). Based on the sequence similarity, the CRISPR repeats are assigned into groups (Kunin et al., 2007). These groups were taken into account in the current classification of CRISPR-Cas systems (Makarova et al., 2011). Although most CRISPR arrays are located on chromosomal DNA, there are examples of CRISPRs located on plasmids (Godde and Bickerton, 2006). In general, CRISPR loci are flanked by A/T rich leader sequences containing...

show abstract

Mechanism of Foreign DNA Selection in a Bacterial Adaptive Immune System

Cited by 231 publications

References 48 publications

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

CRISPR-Cas Systems in Prokaryotes

Contact Info

Product

Resources

About