SUMMARY Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8-nucleotide 5’ sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicates that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.

. Cell. Biol. 8: [4981][4982][4983][4984][4985][4986][4987][4988][4989][4990] 1988), presumably because of the presence of the CKA2 gene. We report here the cloning, sequencing, and disruption of the CKA2 gene. The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A. Saxena, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 7:3409-3417, 1987 (8,13). The beta subunit is required for maximal activity of the catalytic subunit (10) and becomes phosphorylated when the holoenzyme is allowed to undergo autophosphorylation. Although the beta subunit presumably plays a regulatory role, neither the function of this polypeptide nor the significance of autophosphorylation is well understood. The recognition site for casein kinase II consists of a serine or threonine residue located immediately N terminal to a cluster of acidic amino acids (22,25

The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the CKA1 and CKA2 genes, which together are essential for viability. Five independent temperature-sensitive alleles of the CKA2 gene were isolated and used to analyze the function of CKII during the cell cycle. Following a shift to the nonpermissive temperature, cka2 ts strains arrested within a single cell cycle and exhibited a dual arrest phenotype consisting of 50% unbudded and 50% largebudded cells. The unbudded half of the arrested population contained a single nucleus and a single focus of microtubule staining, consistent with arrest in G 1 . Most of the large-budded fraction contained segregated chromatin and an extended spindle, indicative of arrest in anaphase, though a fraction contained an undivided nucleus with a short thick intranuclear spindle, indicative of arrest in G 2 and/or metaphase. Flow cytometry of pheromone-synchronized cells confirmed that CKII is required in G 1 , at a point which must lie at or beyond Start but prior to DNA synthesis. Similar analysis of hydroxyurea-synchronized cells indicated that CKII is not required for completion of previously initiated DNA replication but confirmed that the enzyme is again required for cell cycle progression in G 2 and/or mitosis. These results establish a role for CKII in regulation and/or execution of the eukaryotic cell cycle.Casein kinase II (CKII) 1 is a serine/threonine protein kinase which is ubiquitous among eukaryotic organisms (for review, see Issinger, 1993;Pinna, 1990;Tuazon and Traugh, 1991). The enzyme is composed of a catalytic ␣ and regulatory ␤ subunit that combine to form a native ␣ 2 ␤ 2 holoenzyme which is constitutively active in vitro. How (and indeed whether) the enzyme is regulated in vivo is unknown, though regulation via allosteric effectors (e.g. polyamines), covalent modification, cellular redistribution, and substrate-directed effects have all been proposed. CKII recognizes a Ser of Thr residue followed by a series of acidic residues and phosphorylates a broad and intriguing spectrum of both nuclear and cytoplasmic substrates.Although the physiological role of CKII is not known, several lines of evidence suggest a role for the enzyme in cell prolifer-

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The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non‐permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C‐terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.

We report here the identification of CDC37, which encodes a putative Hsp90 co-chaperone, as a multicopy suppressor of a temperature-sensitive allele (cka2-13 ts ) of the CKA2 gene encoding the ␣ catalytic subunit of protein kinase CKII. Unlike wild-type cells, cka2-13 cells were sensitive to the Hsp90-specific inhibitor geldanamycin, and this sensitivity was suppressed by overexpression of either Hsp90 or Cdc37. However, only CDC37 was capable of suppressing the temperature sensitivity of a cka2-13 strain, implying that Cdc37 is the limiting component. Immunoprecipitation of metabolically labeled Cdc37 from wild-type versus cka2-13 strains revealed that Cdc37 is a physiological substrate of CKII, and Ser-14 and/or Ser-17 were identified as the most likely sites of CKII phosphorylation in vivo. A cdc37-S14,17A strain lacking these phosphorylation sites exhibited severe growth and morphological defects that were partially reversed in a cdc37-S14,17E strain. Reduced CKII activity was observed in both cdc37-S14A and cdc37-S17A mutants at 37°C, and cdc37-S14A or cdc37-S14,17A overexpression was incapable of protecting cka2-13 mutants on media containing geldanamycin. Additionally, CKII activity was elevated in cells arrested at the G 1 and G 2 /M phases of the cell cycle, the same phases during which Cdc37 function is essential. Collectively, these data define a positive feedback loop between CKII and Cdc37. Additional genetic assays demonstrate that this CKII/Cdc37 interaction positively regulates the activity of multiple protein kinases in addition to CKII.Protein kinase CKII is an essential, ubiquitous serine/threonine/tyrosine protein kinase of unknown function. From most sources, the enzyme is composed of two polypeptide subunits, ␣ (35-44 kDa) and ␤ (24 -28 kDa), which combine to form an ␣ 2 ␤ 2 tetramer (for review, see Refs.

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The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer oftopoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vm.. values for the phosphorylation reaction were 0.4 ,.M topoisomerase II and 3.3 ,umol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that posttranslational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA.The topology of DNA has a profound influence on how its genetic information is regenerated, rearranged, and expressed in vivo (1, 2). Consequently, the enzymes that affect the topological structure of nucleic acids play a crucial role in controlling the cellular functions of DNA (1-4). One class of enzymes, the type II topoisomerases, catalyzes changes in the topological state of nucleic acids by passing one intact DNA helix through a transient double-stranded break made in a second helix (5-7). These ubiquitous enzymes are essential for the viability of eukaryotic cells (8)(9)(10) and are involved in many aspects of DNA metabolism (1-4), including replication' (11)(12)(13)(14)(15)(16)(17), repair (18), transcription (13), and chromosome segregation (9,10).Despite the importance of type II topoisomerases to eukaryotic organisms, little information exists concerning the physiological regulation of these enzymes. Although their activity is stimulated >10-fold by cell proliferation (19-21, §), nothing is known about the events that control this increase in activity. Clearly, before the cellular functions of topoisomerase II can be completely described, the factors that regulate its activity must be well understood.Recently, the type I topoisomerase from Novikoff hepatoma cells was shown to exist in vivo as a phosphoprotein (22). Moreover, when purified, the enzyme was found to be sensitive to its state of phosphorylation, with higher levels of incorporation yielding increased rates of activity in vitro (23,24). This suggests that post-translational modification may play a role in the regulation of topoisomerases. With this in mind, the effects of phosphorylation on the activity of topoisomerase II have been examined. This paper describes the interaction between topoisomerase II and casein kinase II from Drosophila melanogaster. While topoisomerase II showed no autophosphorylation under the conditions used, kinase-mediated phosphorylation of the enzyme stimulated its DNA relaxation activity. Furthermore, the effects of phosphorylation could be reversed by treatment with alkaline phosphatase. This study, th...