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[G.sub.1] is a crucial phase of cell growth because the decision to begin another mitotic cycle is made during this period. Occurrence of DNA damage in [G.sub.1] poses a particular challenge, because replication of damaged DNA can be deleterious and because no sister chromatid is present to provide a template for recombinational repair. We therefore have studied the response of Schizosaccharomyces pombe cells to UV irradiation in early [G.sub.1] phase. We find that irradiation results in delayed progression through [G.sub.1], as manifested most critically in the delayed formation of the pre-replication complex. This delay does not have the molecular hallmarks of known checkpoint responses: it is independent of the checkpoint proteins Rad3, Cds1, and Chk1 and does not elicit inhibitory phosphorylation of Cdc2. Irradiated cells eventually progress into S phase and arrest in early S by a rad3--and cds1-dependent mechanism, most likely the intra-S checkpoint. Caffeine alleviates both the intra-[G.sub.1]--and intra-S-phase delays. We suggest that intra-[G.sub.1] delay may be widely conserved and discuss significance and possible mechanisms.

The seqA gene of Escherichia coli controls both the initiation of replication and sequestration in the chromosome by direct interaction with the replication origin oriC. The binding of the protein SeqA to methylated oriC and hemimethylated DNA is strong and specific. SeqA also controls the binding of hemimethylated oriC to other membrane fragments. The initiation of replication is by methylation of oriC and sequestration takes place in the methylated structures formed by replication.

Replication in Escherichia coli starts at a genetically unusual origin, oriC. Multiple oriC copies are found in the rapidly growing cells and the initiation of replication of the origins occurs simultaneously. The sequestration process prevents the starting of a secondary replication and functions by acting on newly-replicated origins.

Complete degradation of individual chromosomes in recombination-deficient recA mutants of Escherichia coli results in asynchrony phenotype. The RecBCD enzyme causes the selective degradation as it is influenced by RecD to a great extent. The unusual separation of daughter nucleotides does not result in asynchrony phenotype. Cells survive in the presence of RecBCD enzyme, which is essential in homologous recombination. Two, four or eight completely duplicated chromosomes are present in the rapidly growing wild-type Escherichia coli when it is treated with rifampin.

The amount of dam methyltransferase in Escherichia coli was measured using an antibody raised against amino acid residues 92 to 106 of the enzyme. The proteins were probed with the antiserum after polyacrylamide gel electrophoresis. Enzyme-linked immunosorbent assay revealed that rapidly growing E. coli cells contain around 130 units of methyltransferases. The enzyme catalyzes the methylation of adenines and has been shown to influence DNA replication.

Abstract Background In many cell types, including the fission yeast Schizosaccharomyces pombe, a set of checkpoints are induced by perturbations of the cell cycle or by DNA damage. Many of the checkpoint responses include a substantial change of the transcriptional pattern. As part of characterising a novel G1/S checkpoint in fission yeast we have investigated whether a transcriptional response is induced after irradiation with ultraviolet light. Results Microarray analyses were used to measure the global transcription levels of all open reading frames of fission yeast after 254 nm ultraviolet irradiation, which is known to induce a G1/S checkpoint. We discovered a surprisingly weak transcriptional response, which is quite unlike the marked changes detected after some other types of treatment and in several other checkpoints. Interestingly, the alterations in gene expression after ultraviolet irradiation were not similar to those observed after ionising radiation or oxidative stress. Pathway analysis suggests that there is little systematic transcriptional response to the irradiation by ultraviolet light, but a marked, coordinated transcriptional response was noted on progression of the cells from G1 to S phase. Conclusion There is little response in fission yeast to ultraviolet light at the transcriptional level. Amongst the genes induced or repressed after ultraviolet irradiation we found none that are likely to be involved in the G1/S checkpoint mechanism, suggesting that the checkpoint is not dependent upon transcriptional regulation.

Abstract Background The fission yeast Schizosaccharomyces pombe is widely-used as a model organism for the study of a broad range of eukaryotic cellular processes such as cell cycle, genome stability and cell morphology. Despite the availability of extensive set of genetic, molecular biological, biochemical and cell biological tools for analysis of protein function in fission yeast, studies are often hampered by the lack of an effective method allowing for the rapid regulation of protein level or protein activity. Results In order to be able to regulate protein function, we have made use of a previous finding that the hormone binding domain of steroid receptors can be used as a regulatory cassette to subject the activity of heterologous proteins to hormonal regulation. The approach is based on fusing the protein of interest to the hormone binding domain (HBD) of the estrogen receptor (ER). The HBD tag will attract the Hsp90 complex, which can render the fusion protein inactive. Upon addition of estradiol the protein is quickly released from the Hsp90 complex and thereby activated. We have tagged and characterised the induction of activity of four different HBD-tagged proteins. Here we show that the tag provided the means to effectively regulate the activity of two of these proteins. Conclusion The estradiol-regulatable hormone binding domain provides a means to regulate the function of some, though not all, fission yeast proteins. This system may result in very quick and reversible activation of the protein of interest. Therefore it will be a powerful tool and it will open experimental approaches in fission yeast that have previously not been possible. Since fission yeast is a widely-used model organism, this will be valuable in many areas of research.

Abstract Background Checkpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired. In fission yeast, checkpoint mechanisms are known to regulate entry into mitosis, but so far no checkpoint inhibiting S phase entry has been identified. Results We have studied the response of germinating Schizosaccharomyces pombe spores to UV irradiation in G1. When germinating spores are irradiated in early G1 phase, entry into S phase is delayed. We argue that the observed delay is caused by two separate mechanisms. The first takes place before entry into S phase, does not depend on the checkpoint proteins Rad3, Cds1 and Chk1 and is independent of Cdc2 phosphorylation. Furthermore, it is not dependent upon inhibiting the Cdc10-dependent transcription required for S phase entry, unlike a G1/S checkpoint described in budding yeast. We show that expression of Cdt1, a protein essential for initiation of DNA replication, is delayed upon UV irradiation. The second part of the delay occurs after entry into S phase and depends on Rad3 and Cds1 and is probably due to the intra-S checkpoint. If the germinating spores are irradiated in late G1, they enter S phase without delay and arrest in S phase, suggesting that the delay we observe upon UV irradiation in early G1 is not caused by nonspecific effects of UV irradiation. Conclusions We have studied the response of germinating S. pombe spores to UV irradiation in G1 and shown that S phase entry is delayed by a mechanism that is different from classical checkpoint responses. Our results point to a mechanism delaying expression of proteins required for S phase entry.