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CHK1 and WEE1 play pivotal roles in G2/M checkpoint following exogenous DNA damage and regulation of DNA replication under normal cellular conditions. Here, we monitored and compared the cell cycle kinetics of mitosis-associated events after CHK1 and WEE1 inhibitor treatments in a human tongue cancer cell line (SAS). A fluorescent ubiquitination-based cell cycle indicator (Fucci) that reflects SCF SKP2 and APC CDH1 E3 ligase activities was used to monitor cell cycle progression. Numerous γH2AX-positive cells were observed within the S phase population of cells following CHK1 inhibitor treatment, and polyploid cells exhibiting DNA damage emerged via abortive mitosis (endomitosis) at 24 h post treatment. While WEE1 inhibitor-treated cells exhibited similar polyploidy via endomitosis at later time points, they possessed fewer γH2AX foci during S phase, and polyploid cells exhibiting DNA damage were scarce. Instead, mitosis duration greatly extended and was accompanied by an abnormal emission of Fucci red fluorescence. Kinetic analysis of Fucci fluorescence revealed that abnormal emission occurred at early M phase in a manner independent of green fluorescence degradation as a marker of APC CDH1 activation. When an inhibitor of the essential spindle checkpoint factor MPS1 was co-treated with a WEE1 inhibitor, the elongated mitosis duration and abnormal red fluorescence were abrogated, and WEE1-induced reduction of clonogenic survival was offset. We demonstrate novel differential effects on mitosis-associated events following CHK1 and WEE1 inhibitor treatments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mek1 is a Chk2/Rad53/Cds1-related protein kinase that is required for proper meiotic progression of Schizosaccharomyces pombe. However, the molecular mechanisms of Mek1 regulation and Mek1 phosphorylation targets are unclear. Here, we report that Mek1 is phosphorylated at serine-12 (S12), S14 and threonine-15 (T15) by Rad3 (ATR) and/or Tel1 (ATM) kinases that are activated by meiotic programmed double-strand breaks (DSBs). Mutations of these sites by alanine replacement caused abnormal meiotic progression and recombination rates. Phosphorylation of these sites triggers autophosphorylation of Mek1; indeed, alanine replacement mutations of Mek1-T318 and -T322 residues in the activation loop of Mek1 reduced Mek1 kinase activity and meiotic recombination rates. Substrates of Mek1 include Mus81-T275, Rdh54-T6 and Rdh54-T673. Mus81-T275 is known to regulate the Mus81 function in DNA cleavage, whereas Rdh54-T6A/T673A mutant cells showed abnormal meiotic recombination. Taken together, we conclude that the phosphorylation of Mek1 by Rad3 or Tel1, Mek1 autophosphorylation and Mus81 or Rdh54 phosphorylation by Mek1 regulate meiotic progression in S. pombe.

The meiosis-specific mug28(+) gene of Schizosaccharomyces pombe encodes a putative RNA-binding protein with three RNA recognition motifs (RRMs). Live observations of meiotic cells that express Mug28 tagged with green fluorescent protein (GFP) revealed that Mug28 is localized in the cytoplasm, and accumulates around the nucleus from metaphase I to anaphase II. Disruption of mug28(+) generated spores with low viability, due to the aberrant formation of the forespore membrane (FSM). Visualization of the FSM in living cells expressing GFP-tagged Psy1, an FSM protein, indicated that mug28Delta cells harbored abnormal FSMs that contained buds, and had a delayed disappearance of Meu14, a leading edge protein. Electron microscopic observation revealed that FSM formation was abnormal in mug28Delta cells, showing bifurcated spore walls that were thicker than the nonbifurcated spore walls of the wild type. Analysis of Mug28 mutants revealed that RRM3, in particular phenylalanin-466, is of primary importance for the proper localization of Mug28, spore viability, and FSM formation. Together, we conclude that Mug28 is essential for the proper maturation of the FSM and the spore wall.

We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Delta cells are similar to those of meu13Delta cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Delta cells is not so conspicuous as meu13Delta cells, and no meiotic delay is observed in mcp7Deltameu13Delta cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Delta cells, whereas Meu13 becomes less stable in mcp7Delta cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination.

Human Lats2, a novel serine/threonine kinase, is a member of the Lats kinase family that includes the Drosophila tumour suppressor lats/warts. Lats1, a counterpart of Lats2, is phosphorylated in mitosis and localized to the mitotic apparatus. However, the regulation, function and intracellular distribution of Lats2 remain unclear. Here, we show that Lats2 is a novel phosphorylation target of Aurora-A kinase. We first showed that the phosphorylated residue of Lats2 is S83 in vitro. Antibody that recognizes this phosphorylated S83 indicated that the phosphorylation also occurs in vivo. We found that Lats2 transiently interacts with Aurora-A, and that Lats2 and Aurora-A co-localize at the centrosomes during the cell cycle. Furthermore, we showed that the inhibition of Aurora-A-induced phosphorylation of S83 on Lats2 partially perturbed its centrosomal localization. On the basis of these observations, we conclude that S83 of Lats2 is a phosphorylation target of Aurora-A and this phosphorylation plays a role of the centrosomal localization of Lats2.

Mitogen-dependent progression through the first gap phase (G1) of the mammalian cell-division cycle is precisely regulated so that normal cell division is coordinated with cell growth, while the initiation of DNA synthesis (S phase) is precisely ordered to prevent inappropriate amplification of the DNA that may cause genome instability. To ensure that these fundamental requirements of cell division are met, cells have developed a surveillance mechanism based on an intricate network of protein kinase signaling pathways that lead to several different types of checkpoints. Since these checkpoints are central to the maintenance of the genomic integrity and basic viability of the cells, defects in these pathways may result in either tumorigenesis or apoptosis, depending on the severity and nature of the defects. This review summarizes the genetic and molecular mechanisms of checkpoint activation in the G1/S and S phases of the mammalian cell cycle that monitor DNA damage and replication. The relevance of these mechanisms to the origin of cancer is also discussed.

Using a meiosis-specific subtracted cDNA library of Schizosaccharomyces pombe, we identified meu14+ as a gene whose expression is upregulated during meiosis. Transcription of meu14+ is induced abruptly after the cell enters meiosis. Its transcription is dependent on the meiosis-specific transcription factor Mei4. In meu14Delta cells, the segregation and modification of the SPBs (spindle pole bodies) and microtubule elongation during meiosis II were aberrant. Meiotic meu14Delta cells consequently produced a high frequency of abnormal tetranucleate cells harboring aberrant forespore membranes and failed to produce asci. In wild-type cells harboring the integrated meu14+-gfp fusion gene, Meu14-GFP first appeared inside the nuclear region at prophase II, after which it accumulated beside the two SPBs at metaphase II. Thereafter, it formed two ring-shaped structures that surrounded the nucleus at early anaphase II. At post-anaphase II, it disappeared. Meu14-GFP appears to localize at the border of the forespore membrane that later develops into spore walls at the end of sporulation. This was confirmed by coexpressing Spo3-HA, a component of the forespore membrane, with Meu14-GFP. Taken together, we conclude that meu14+ is crucial in meiosis in that it participates in both the nuclear division during meiosis II and the accurate formation of the forespore membrane.

Background: The Mcm proteins are a family of six homologous proteins (Mcm2-7) that play an important role in DNA replication. They form Mcm4/6/7 and Mcm2/4/6/7 complexes, but their structures are not known., Results: We found that the human Mcm2/4/6/7 tetramer forms a toroidal structure, with a central cavity about 3-4 nm in diameter. Observations were made using electron microscopy, employing the image analysis of single particles. The most predominant averaged image displayed a toroid harbouring four bulges forming corners, one of which was larger than the others. This structure was very similar to the mouse Mcm2/4/6/7 tetramer that was independently prepared and analysed by electron microscopy. These toroidal structures are distinct from that of the Mcm4/6/7 hexamer, which was also examined by electron microscopy. GST(glutathione S-transferase)-pull down and two hybrid experiments suggest that a putative Mcm6-Mcm6 hinge contributes to the formation of the Mcm7/4/6/6/4/7 heterohexamer., Conclusions: The Mcm2/4/6/7 tetramer forms a toroidal structure that is distinct from that of the Mcm4/6/7 hexamer in size and shape.

During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression.

Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.

Kcc4, a kinase of the budding yeast Saccharomyces cerevisiae, is homologous to the bud neck protein kinases Hsl1/Nik1 and Gin4. We report here that a GFP-Kcc4 fusion protein is localized at the bud neck and that the non-kinase domain is required for this localization. We also demonstrate that Kcc4 associates with septin proteins in vitro and in vivo by two-hybrid analysis, GST pull-down experiments, immunoprecipitation, and analysis of direct association with affinity-purified GST-Kcc4 and MBP-Septin proteins. From the results obtained here, we suggest that Cdc11 is the primary association partner of Kcc4.

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity., (Copyright 2000 Academic Press.)

The rad24(+) gene of Schizosaccharomyces pombe encodes a ubiquitously expressed 14-3-3 protein. We report here that Deltarad24 cells displayed a defect in diploid colony formation, although they conjugated efficiently. We found that a cumulative deletion of mei2(+) gene almost completely suppressed this defect, and demonstrated using two-hybrid analysis that Rad24 protein directly associates with Mei2 protein by recognizing Ser-438 which is a phosphorylation target of Pat1 kinase. We conclude that constitutive progression to meiosis, caused by lack of Mei2 inhibition due to the absence of Rad24 protein, is the primary cause of the proliferative deficiency observed in Deltarad24 cells.

Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle. Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary. In this study, we examined the transcriptional activities of isolated human MCM gene promoters. Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression. In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation. Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells. Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters.

The human embryonal carcinoma cells NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N, N'-hexamethylene-bis-acetamide and cease to grow in several days. Transcription factors of the E2F/DP family have been shown to be closely related to the regulation of cell proliferation. To analyse cellular proteins which interact with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lambdaZAP II NEC14 cell library with the 32P-labeled GST (Glutathione S-transferase)-E2F-1 fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix-loop-helix (HLH) motif, but lacks the basic domain and the zipper structure usually found at N- and C-terminal sides to the HLH motif, respectively. The arrangement of amino acids in the helix 1 and helix 2 regions is quite similar to those of Mxi and Mad, but different from those of E2F-1 and DP-1. Western blot analysis of the immunoprecipitates prepared with anti-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP-1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1. Although E2FBP1 lacks the transactivation domain, it stimulates E2F site-dependent transcription in cooperation with E2F-1.

We report here the comparative analysis of human Mcm/P1 proteins (HsMcm2, -3, -5 and -7), including a characterization of their mutual interactions, cell cycle dependent expression and nuclear localization during the cell cycle and the quiescent state. The mRNA levels of these genes, which undergo cell cycle dependent oscillations with a peak at G1/S phase, may be regulated by E2F motifs, two of which were detected in the 5' upstream region of the HsMCM5 gene. In contrast, the protein levels of these Mcm proteins were found to remain rather constant during the HeLa cell cycle. However, their levels gradually increased in a variable manner as KD cells progressed from GO into the G1/S phase. In the GO stage, the amounts of HsMcm2 and -5 proteins were much lower than those of HsMcm7 and -3 proteins, suggesting that they are not present in stoichiometric amounts, and that only a proportion of these molecules actively participate in cell cycle regulation as part of Mcm/P1 complexes.

In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re-association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.

Background: The tight regulatory mechanism that prevents more than one round of chromosomal DNA replication per cell cycle is thought to require the function of Mcm/P1 proteins. We report here the structural and functional analyses of HsMcm6, a human homologue of the Mis5 of Schizosaccharomyces pombe., Results: We demonstrate here that the transcription of the HsMCM6 gene was repressed in quiescent cells but was rapidly induced at the G1/S phase by growth factor stimulation. The 5' regulatory region of the HsMCM6 gene was found to harbour four putative E2F binding motifs, and these were responsible for the promoter activity. The HsMcm6 protein level oscillated during the cell cycle, with a peak at the G1/S phase. We also showed that the cell-cycle dependent change of subcellular localization of HsMcm6 resembles those of other Mcm/P1 proteins. HsMcm6 consists of two forms, a form extractable by Nonidet P-40 and the nucleus-bound form. A demonstration of the association of HsMcm6 with HsMcm2 and HsMcm7 in vivo supports the idea that they behave as a heteromeric complex. We mapped the HsMCM6 gene at 2q12-14., Conclusion: The results indicate that the behaviour of HsMcm6 is reminiscent of replication licensing factor like other Mcm/P1 family members.

Nitric oxide-generating vasodilators inhibit vascular smooth muscle cell proliferation. To elucidate the mechanism underlying this process, we investigated the effect of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide-releasing agent, on the smooth muscle cell cycle. When G0 cells were stimulated with fetal bovine serum and basic fibroblast growth factor, DNA synthesis assessed by [3H]thymidine incorporation started about 15 h later. SNAP dose-dependently inhibited this incorporation, and this effect was maximal at 100 microM. This inhibition was attenuated when SNAP was added after 9-12 h. SNAP inhibited the activity of cyclin-dependent kinase 2 (Cdk2) and phosphorylation of the retinoblastoma protein, both of which usually increased from about 9 h, whereas it did not inhibit the activities of cyclin D-associated kinase(s), Cdk4, and Cdk6, which normally increased from 0-3 h. Although SNAP reduced the mRNA levels of cyclins E and A, it neither reduced their protein levels nor impaired their association with Cdk2. SNAP did not reduce the mRNA levels of cyclins G, C, and D1, Cdk2, Cdk4, and Cdk5, which were normally elevated from 0-3 h. The mRNA and protein levels of the Cdk inhibitor p21 were high in the early G1 phase, peaking at 3 h and then rapidly decreasing after 6 h. In the presence of SNAP, however, p21 expression was enhanced, and moreover, the later decrease disappeared. SNAP also increased the amount of Cdk2-associated p21. These results suggested that nitric oxide inhibits the G1/S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that p21 induction is involved in the Cdk2 inhibition.

Replication licensing factor is thought to be involved in the strict control of the initiation of DNA replication in eukaryotes. We identified a 100 kDa protein as a candidate for the licensing factor in Xenopus egg extracts. This protein was required for replication; it bound to sperm DNA before the formation of nuclei and apparently dissociated from the nuclear DNA during the progression of replication without being transported into the nuclei. An immunologically homologous protein in HeLa cells behaved similarly to the Xenopus protein during the cell cycle. Cloning and sequencing of the cDNAs encoding the Xenopus and human proteins revealed that they are homologs of yeast Mcm3, a putative yeast DNA replication licensing factor.

We cloned by phenotypic complementation a novel Saccharomyces cerevisiae's multicopy suppressor of the Schizosaccharomyces pombe cdc10-129 mutant which we call HAC1, an acronym of 'homologous to ATF/CREB 1'. It encodes a bZIP (basic-leucine zipper) protein of 230 amino acids with close homology to the mammalian ATF/CREB transcription factor and gel-retardation assays showed that it binds specifically to the CRE motif. HAC1 is not essential for viability. However, the hac1 disruptant becomes caffeine sensitive, which is suppressed by multicopy expression of the yeast PDE2 (Phosphodiesterase 2) gene. Although the mRNA level of HAC1 is almost constitutive throughout the cell cycle, it fluctuates during meiosis. The upstream region of the HAC1 gene contains a T4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes. These results suggest that HAC1 may also be one of the meiotic genes.

In the fission yeast Schizosaccharomyces pombe, meiosis is initiated by the action of Mei2 in a complex cascade activated following conjugation. We have isolated a new gene named rep1+ that is required for the initiation of premeiotic DNA synthesis. rep1+ encodes a 53 kDa protein with one zinc finger motif that is essential for function, and effectively rescues a null mutant of the res1+ gene but only partially a temperature-sensitive mutant of the cdc10+ gene, both of which are required for the onset of mitotic, as well as premeiotic, S phase. Deletion of rep1+ has no apparent effects on the mitotic cell cycle or conjugation, but blocks the initiation of premeiotic DNA synthesis. However, this defect is partially suppressed when rapidly growing cells are induced to conjugate, indicating that the rep1+ function is at least partly substituted by those of the genes controlling the 'start' of the mitotic cell cycle. The rep1 null mutant fails to induce the res2+ gene, a newly identified res1+ homolog cooperating with Cdc10 and acting for the onset of mitotic and premeiotic DNA synthesis, as well as for meiotic division. The rep1+ gene itself is induced moderately during nitrogen starvation but highly during conjugation, and this induction is dependent on both ste11+ and mating pheromones but independent of mei2+. Thus, rep1+ controls the initiation of premeiotic DNA synthesis via induction and/or activation of Res2 and some other essential factors in a cascade independent of Mei2.

The differential activation of cyclin and cyclin-dependent kinase genes by the adenovirus E1A gene product (E1A) or serum factors was studied with a rat 3Y1 derivative cell line, g12-21, in which the E1A12S cDNA can be expressed in response to dexamethasone (dex). The induction of DNA synthesis in quiescent g12-21 cells occurred within 12 h after serum stimulation, while it occurred within 8 h after treatment with dex. The expression of cyclin D1 and E genes in the serum-stimulated cells was induced in mid G1 and mid to late G1, respectively, while that of the cyclin D1 gene was not induced and the induction of the cyclin E gene was shifted to the G1/S boundary in the dex-treated cells. The cdk2 gene was induced in late G1 and cdc2 and cyclin A genes at the G1/S boundary in both serum-stimulated and dex-treated cells. These results suggest that E1A skips cell cycle events which normally occur in early to mid G1 and may directly activate late-response genes. Analysis of the transcription factor E2F complexes formed in the promoter regions of cdc2 and dihydrofolate reductase genes showed that the amount of complexes formed is maximal at the G1/S boundary, but decreases in S phase when these genes are transcribed extensively.

We have isolated a new cell division cycle gene (res1+) required for entry into S phase, as a multicopy dual suppressor of the pat1 and cdc10 mutants of the fission yeast Schizosaccharomyces pombe. The res1+ gene specifies a 72 kDa protein with two copies of the cdc10/SWI6 motif. A disruptant of res1+ grows poorly at 30 degrees C with severe heat- and cold-sensitivities, and completely arrests in G1 at 36 degrees C and 23 degrees C. The arrested disruptant retains a full conjugation ability. In addition to the cdc10/SWI6 motif, Res1 and SWI4 proteins share a remarkable homology in their amino-terminal region, whereas Cdc10 and SWI6 do so in their carboxy-terminal region. Moreover, the amino-terminal region is essential for the function of Res1 as it is for the function of SWI4. Furthermore, analogous to the relationship of SWI4 to SWI6, the res1+ gene effectively rescues cdc10 mutants, but the cdc10+ gene cannot rescue the res1- phenotype. Thus, striking similarities exist in both structural and functional relationships between Res1 and SWI4, and between Cdc10 and SWI6. In view of the fact that SWI4 and SWI6 form a transcription factor complex and activate promoters containing the SWI4/SWI6 dependent cell-cycle box, Res1 might be a putative association partner of Cdc10 which appears to be involved at least in the activation of promoters containing a MluI cell-cycle box.