No abstract

Cell cycle progression is dependent on the sequential activity of cyclin‐dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK‐activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7–cyclin H (Mcs6–Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6–Mcs2 CAK defining Csk1 as a CAK‐activating kinase (CAKAK).

The cyclin‐dependent kinase (CDK)‐activating kinase, CAK, from mammals and amphibians consists of MO15/CDK7 and cyclin H, a complex which has been identified also as a RNA polymerase II C‐terminal domain (CTD) kinase. While the Schizosaccharomyces pombe cdc2 gene product also requires an activating phosphorylation, the enzyme responsible has not been identified. We have isolated an essential S.pombe gene, mop1, whose product is closely related to MO15 and to Saccharomyces cerevisiae Kin28. The functional similarity of Mop1 and MO15 is reflected in the ability of MO15 to rescue a mop1 null allele. This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H‐related cyclin Mcs2 of S.pombe. Also, Mop1 and Mcs2 can associate with the heterologous partners human cyclin H and MO15, respectively. Moreover, the rescue of a temperature‐sensitive mcs2 strain by expression of mop1+ demonstrates a genetic interaction between mop1 and mcs2. In a functional assay, immunoprecipitated Mop1‐Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. The CAK activity of Mop1‐Mcs2 distinguishes it from the related CDK‐cyclin pair Kin28‐Ccl1 from S.cerevisiae, and supports the notion that Mop1‐Mcs2 may represent a homolog of MO15‐cyclin H in S.pombe with apparent dual roles as a RNA polymerase CTD kinase and as a CAK.

A series of high affinity second-generation thiazolopiperidine inhibitors of PI3Kγ were designed based on some general observations around lipid kinase structure. Optimization of the alkylimidazole group led to inhibitors with higher levels of PI3Kγ selectivity. Additional insights into PI3K isoform selectivity related to sequence differences in a known distal hydrophobic pocket are also described.

The mutD (dnaQ) gene of Escherichia coli codes for the proofreading activity of DNA polymerase III. The very strong mutator phenotype of mutD5 strains seems to indicate that their postreplicational mismatch repair activity is also impaired. We show that the mismatch repair system of mutDS strains is functional but saturated, presumably by the excess of DNA replication errors, since it is recovered by inhibiting chromosomal DNA replication. This recovery depends on de novo protein synthesis.The mechanisms ensuring accuracy of DNA replication have been elucidated by study of mutator mutants affecting specific accuracy functions (for a review, see reference 4). In Escherichia coli, mutator mutations mapping in the dnaE gene presumably affect nucleotide selection by DNA polymerase III (28). Mutations mapping in the dnaQ gene (also called mutD) affect the proofreading 3'->5' exonuclease function of the DNA polymerase III holoenzyme (6, 8), whereas mutH, mutL, mutS, and mutU mutations cause defects in methyl-directed postreplicative mismatch correction (for reviews, see references 21 and 24). mutD5 and dnaQ49 mutants are the most potent mutator strains known. All classes of transition, transversion, and frameshift mutations are increased up to 105-fold when mutD5 strains are grown in rich medium (5, 10, 11). The mutD5 gene of E. coli encodes the epsilon subunit, which carries out the 3'-*5' proofreading exonuclease function of the DNA polymerase III holoenzyme (6,8). However, the high-mutation-rate effect of mutD5 strains cannot be explained only by the defect in exonuclease activity, as this mutation rate is comparable to the error rate of in vitro replication with a polymerase devoid of proofreading activity (9, 16). The strong mutator phenotype of mutD5 suggested that mismatch repair functions may be impaired as well. This prediction was supported by results from DNA heteroduplex transfection experiments (23,25,26). One hypothesis is that the high error rate in DNA replication saturates mismatch repair in mutD5 mutants (23). Therefore, we have tested the mismatch repair capacity of mutD5 strains under conditions in which chromosomal DNA replication was or was not allowed. Mismatch repair activity was determined by the extent of pure infective centers derived from infection with packaged hemimethylated heteroduplexes of lambda DNA. Under these conditions, bacteria proficient in mismatch repair produced essentially pure infective centers, whereas bacteria deficient in mismatch repair produced mostly mixed infective centers.To evaluate mismatch repair activity, pure hemimethy-* Corresponding author. t Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403. lated heteroduplexes of lambda DNA containing an A. C or a G T mismatch were artificially constructed and introduced into mutD5 and other appropriate strains. Hemimethylated heteroduplexes of lambda DNA with defined mismatches were prepared as previously described (19) by reannealing the separated DNA strands of a lambda mutant carry...