We have isolated fission yeast mutants that enter mitosis when DNA replication is blocked with hydroxyurea. The mutants define eight linkage groups, three of which consist of alleles of the radl, rad3, and radl 7 genes. Recently, these fission yeast genes have been shown to be required for radiation-induced cell cycle arrest, as is the budding yeast RAD9 gene. The other five genes are called bus (hydroxy_urea sensitive) 1-5. We propose that these genes participate in an intraceUular signal transduction pathway that monitors the completion of DNA replication and transmits information to the mitotic control protein cdc2. Mutations that bypass the requirement for cdc25 (an activator of the mitotic regulator cdc2) also uncouple mitosis from DNA replication. However, mitosis is blocked by inhibitors of DNA replication in strains in which the cdc25 gene has been deleted, indicating that although cdc25 influences the coupling of mitosis to the completion of DNA replication, it is not essential for this control.[Key Words: Cell cycle; checkpoint; radiation; cdc25; S. pombe] Received May 4, 1992; revised version accepted August 27, 1992.A normal cell cycle consists of a temporally ordered series of events. This order is maintained partly by mechanisms that can monitor completion of earlier events, called "checkpoint" (Hartwell and Weinert 1989) or "feedback" (Murray and Kirschner 1989)controls. Elimination of such controls by mutation or chemical treatment disrupts the dependencies characteristic of ordered progression through the cell cycle (for review, see Hartwell and Weinert 1989). Although mutations that abolish several different cell cycle checkpoints have been identified (Weinert and Hartwell 1988;Hoyt et al. 1991;Li and Murray 1991), it is not clear how these controls work. Early steps must "signal" late steps, but little is known about what these signals are or how they are monitored.We are investigating this problem by studying the coupling of mitosis to the completion of DNA replication in the fission yeast Schizosaccharomyces pombe. This sys-

Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. In most species this is accomplished by preventing activation of the cell-division kinase Cdc2, which regulates entry into mitosis. The Chk1 kinase, an effector of the DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2. Phosphorylation of Cdc25 promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2. Here we propose that a similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the replication checkpoint) in fission yeast. We show by mutagenesis that Chk1 functions redundantly with the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. We conclude that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint response to unreplicated DNA.

of nucleotides in the cell are known to block DNA Department of Genetics, Harvard Medical School, 200 Longwood elongation (Vassilev and Russev, 1984; Friedberg et al Hartwell, 1995;Longhese et al., 1996). Here we rqh1 mutants. rqh1 ⍣ , previously known as hus2 ⍣ , demonstrate that reversible S phase arrest also requires encodes a putative DNA helicase related to the Escherprotective functions that are distinct from cell cycle ichia coli RecQ helicase, with particular homology to checkpoint controls. the gene products of the human BLM and WRN genesWe have previously studied S phase arrest in the fission and the Saccharomyces cerevisiae SGS1 gene. BLM and yeast Schizosaccharomyces pombe, by isolating mutants WRN are mutated in patients with Bloom's syndrome that are sensitive to hydroxyurea (HU), a drug that blocks and Werner's syndrome respectively. Both syndromes DNA replication by depleting deoxynucleotides. Our are associated with genomic instability and cancer screening strategy was based on the observation that susceptibility. We show that, like BLM and SGS1, checkpoint-defective cells undergo an aberrant mitosis rqh1 ⍣ is required to prevent recombination and that ('cut') when treated with HU (Enoch and Nurse, 1990). in fission yeast suppression of inappropriate recombinUnder the same conditions normal cells cease DNA ation is essential for reversible S phase arrest.synthesis and arrest cell division, displaying an elongated Keywords: cell cycle/hus2/recombination/RecQ DNA cell morphology. By screening for mutants that 'cut' in helicase/Schizosaccharomyces pombe HU, a number of checkpoint-defective HU-sensitive (hus) mutants were identified (Enoch et al., 1992). Several of the mutants were found to be allelic to previously known

No abstract

beta-Interferon (beta-IFN) gene expression can be induced by poly(I)-poly(C) or virus, but there is considerable variation in the extent of induction between different cell lines. We characterized two poorly inducible human cell lines, HeLa and 143 thymidine kinase negative (143 tk-), to define cellular factors involved in the activation of the beta-IFN gene. We show that the deficiency in beta-IFN induction in these cells can be complemented by fusion to highly inducible mouse cells. We conclude that the human cells are deficient in a trans-acting factor required for B-IFN gene activation. The level of induction of the beta-IFN gene in HeLa and 143 tk- cells can also be increased by priming with IFN before induction. If IFN priming is carried out in the presence of cycloheximide, a approximately 200-fold increase in induction is observed. We conclude that activation of the beta-IFN gene requires an IFN-inducible factor that is only expressed at low levels in unprimed HeLa and 143 tk- cells.