The number and diversity of CRISPR-Cas systems has substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that occurred since the publication of the latest classification in 2015. The new classification includes 2 classes, 6 types and 33 subtypes compared to 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems that now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPRlinked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas. This work complements Ref. 34 by experimentally validating the prediction made in Ref. 33, that interference-deficient subtype IF CRISPR-Cas systems encoded in Tn7like transposons enable crRNA-dependent transposition.

Type III CRISPR-Cas systems in prokaryotes provide immunity against invading nucleic acids through the coordinated degradation of transcriptionally active DNA and its transcripts by the Csm effector complex. The Cas10 subunit of the complex contains an HD nuclease domain that is responsible for DNA degradation and two Palm domains with elusive functions. In addition, Csm6, a ribonuclease that is not part of the complex, is also required to provide full immunity. We show here that target RNA binding by the Csm effector complex of triggers Cas10 to synthesize cyclic oligoadenylates (cA ; = 2 to 6) by means of the Palm domains. Acting as signaling molecules, cyclic oligoadenylates bind Csm6 to activate its nonspecific RNA degradation. This cyclic oligoadenylate-based signaling pathway coordinates different components of CRISPR-Cas to prevent phage infection and propagation.

Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA.

Streptococcus thermophilus (St) type III-A CRISPR-Cas system restricts MS2 RNA phage and cuts RNA in vitro. However, the CRISPR array spacers match DNA phages, raising the question: does the St CRISPR-Cas system provide immunity by erasing phage mRNA or/and by eliminating invading DNA? We show that it does both. We find that (1) base-pairing between crRNA and target RNA activates single-stranded DNA (ssDNA) degradation by StCsm; (2) ssDNase activity is confined to the HD-domain of Cas10; (3) target RNA cleavage by the Csm3 RNase suppresses Cas10 DNase activity, ensuring temporal control of DNA degradation; and (4) base-pairing between crRNA 5'-handle and target RNA 3'-flanking sequence inhibits Cas10 ssDNase to prevent self-targeting. We propose that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phage DNA, but not the host DNA.

We present a measure for the evaluation of secondary structure prediction methods that is based on secondary structure segments rather than individual residues. The algorithm is an extension of the segment overlap measure Sov, originally defined by Rost et al. (J Mol Biol 1994;235:13-26). The new definition of Sov corrects the normalization procedure and improves Sov's ability to discriminate between similar and dissimilar segment distributions. The method has been comprehensively tested during the second Critical Assessment of Techniques for Protein Structure Prediction (CASP2). Here, we describe the underlying concepts , modifications to the original definition, and their significance. Proteins 1999;34:220-223. Published 1999 Wiley-Liss, Inc. †

The repair of damaged DNA is coupled to the completion of DNA replication by several cell cycle checkpoint proteins, including, for example, in fission yeast Rad1(Sp), Hus1(Sp), Rad9(Sp) and Rad17(Sp). We have found that these four proteins are conserved with protein sequences throughout eukaryotic evolution. Using computational techniques, including fold recognition, comparative modeling and generalized sequence profiles, we have made high confidence structure predictions for the each of the Rad1, Hus1 and Rad9 protein families (Rad17(Sc), Mec3(Sc) and Ddc1(Sc) in budding yeast, respectively). Each of these families was found to share a common protein fold with that of PCNA, the sliding clamp protein that tethers DNA polymerase to its template. We used previously reported genetic and biochemical data for these proteins from yeast and human cells to predict a heterotrimeric PCNA-like ring structure for the functional Rad1/Rad9/Hus1 complex and to determine their exact order within it. In addition, for each individual protein family, contact regions with neighbors within the PCNA-like ring were identified. Based on a molecular model for Rad17(Sp), we concluded that members of this family, similar to the subunits of the RFC clamp-loading complex, are capable of coupling ATP binding with conformational changes required to load a sliding clamp onto DNA. This model substantiates previous findings regarding the behavior of Rad17 family proteins upon DNA damage and within the RFC complex of clamp-loading proteins.

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA′) and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA′ and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA′, imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA′-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance. (1), a Cfamily DNA polymerase implicated in error-prone bypass of DNA lesions. Loss of DnaE2 activity renders Mtb hypersensitive to DNA damage and eliminates induced mutagenesis. Moreover, dnaE2 deletion attenuates virulence and reduces the frequency of drug resistance in vivo. Mtb contains two DnaE-type polymerases; the other, DnaE1, provides essential, high-fidelity replicative polymerase function (1). However, the basis for the functional specialization of the DnaE subunits remains unclear (2, 3). Although structural determinants such as active-site architecture contribute significantly to inherent fidelity, it is possible that differential interactions with other DNA metabolic proteins modulate polymerase function.Bacterial genomes containing a DnaE2-type DNA polymerase almost invariably encode a homolog of ImuB (4-6), a putative Yfamily polymerase that is usually present in a LexA-regulated imuA-imuB-dnaE2 gene cassette (5). In Caulobacter crescentus, both ImuB and ImuA are required for induced mutagenesis and damage tolerance (6) whereas plasmid-encoded DnaE2 and ImuB mediate UV-induced mutagenesis in Deinococcus deserti (7). Although distributed widely across the bacterial domain, the imuA-imuB-dnaE2 cassette is not found in organisms possessing umuDC homologs (5). This suggests that the encoded proteins perform an analogous function to DNA polymerase V (8), the Y-family member required for damage-induced mutagenesis in Escherichia coli (9).Mtb contains a putative SOS-inducible operon, Rv3395c-Rv3394c (1, 10), locat...

The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295-350 of hMYH and to residues 245-293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH-hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.