Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems

Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant 5R01DK097768-03)National Institutes of Health (U.S.) (Grant GM10407

Viral genome packaging terminase cleaves DNA using the canonical RuvC-like two-metal catalysis mechanism

Bacteriophages and large dsDNA viruses encode sophisticated machinery to translocate their DNA into a preformed empty capsid. An essential part of this machine, the large terminase protein, processes viral DNA into constituent units utilizing its nuclease activity. Crystal structures of the large terminase nuclease from the thermophilic bacteriophage G20c show that it is most similar to the RuvC family of the RNase H-like endonucleases. Like RuvC proteins, the nuclease requires either Mn2+, Mg2+ or Co2+ ions for activity, but is inactive with Zn2+ and Ca2+. High resolution crystal structures of complexes with different metals reveal that in the absence of DNA, only one catalytic metal ion is accommodated in the active site. Binding of the second metal ion may be facilitated by conformational variability, which enables the two catalytic aspartic acids to be brought closer to each other. Structural comparison indicates that in common with the RuvC family, the location of the two catalytic metals differs from other members of the RNase H family. In contrast to a recently proposed mechanism, the available data do not support binding of the two metals at an ultra-short interatomic distance. Thus we postulate that viral terminases cleave DNA by the canonical RuvC-like mechanism

A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing

Gp39, a small protein encoded by Thermus thermophilus phage P23–45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity

Transcription regulation of the EcoRV restriction–modification system

When a plasmid containing restriction–modification (R–M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R–M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R–M systems, specialized Control (C) proteins coordinate expression of the R and the M genes. C proteins bind to DNA sequences called C-boxes and activate expression of their cognate R genes and inhibit the M gene expression, however the mechanisms remain undefined. Here, we studied the regulation of gene expression in the C protein-dependent EcoRV system. We map the divergent EcoRV M and R gene promoters and we define the site of C protein-binding that is sufficient for activation of the EcoRV R transcription

Structural Basis for Polθ-Helicase DNA Binding and Microhomology-Mediated End-Joining

DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Polθ) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Polθ is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Polθ-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Polθ helicase domain (Polθ-hel) in complex with DNA containing different 3\u27-ssDNA overhangs. The structures reveal the sequential conformations adopted by Polθ-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Polθ-hel subdomains and its functional dimeric state are pivotal for aligning the 3\u27-ssDNA overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Polθ-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Polθ-hel

tRNA Anticodon Cleavage by Target-Activated CRISPR-Cas13a Effector

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA–guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease

Modular architecture of nucleotide-binding pockets

Recently, modularity has emerged as a general attribute of complex biological systems. This is probably because modular systems lend themselves readily to optimization via random mutation followed by natural selection. Although they are not traditionally considered to evolve by this process, biological ligands are also modular, being composed of recurring chemical fragments, and moreover they exhibit similarities reminiscent of mutations (e.g. the few atoms differentiating adenine and guanine). Many ligands are also promiscuous in the sense that they bind to many different protein folds. Here, we investigated whether ligand chemical modularity is reflected in an underlying modularity of binding sites across unrelated proteins. We chose nucleotides as paradigmatic ligands, because they can be described as composed of well-defined fragments (nucleobase, ribose and phosphates) and are quite abundant both in nature and in protein structure databases. We found that nucleotide-binding sites do indeed show a modular organization and are composed of fragment-specific protein structural motifs, which parallel the modular structure of their ligands. Through an analysis of the distribution of these motifs in different proteins and in different folds, we discuss the evolutionary implications of these findings and argue that the structural features we observed can arise both as a result of divergence from a common ancestor or convergent evolution

Tail-Tape-Fused Virion and Non-Virion RNA Polymerases of a Thermophilic Virus with an Extremely Long Tail

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species

Parg Is Essential for Polθ-Mediated Dna End-Joining by Removing Repressive Poly-Adp-Ribose Marks

DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1- independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ