DPBII, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(E) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPBII gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption ofDPBII is lethal, indicating that DPBII is essential for cell proliferation. In thermosensitive dpbll-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability. dpbll-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpbll cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpbll is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathwaj during the S phase of the cell cycle to sense stalled DNA replication.Chromosomal DNA is accurately replicated only once in S phase of the cell cycle. In Saccharomyces cerevisiae, if DNA replication is blocked or DNA is damaged by reagents or aberrant DNA synthesis, a checkpoint system arrests the cell cycle (for review, see refs. 1 and 2). The DNA damage checkpoint requires the RAD9, RAD17, RAD24, MEC1, MEC2/ RAD53/SADl/SPKI, and MEC3 genes (3-6). The S-phase checkpoint that finds the replication block partially overlaps the DNA damage checkpoint, as MEC1 and MEC2 are required for both checkpoints (5,6).A checkpoint at the S-phase onset has been also described in the fission yeast Schizosaccharomycespombe (for review, see ref. 7). cdc18, rad4/cut5, and cdtl mutations prevent initiation of DNA synthesis and allow cells to enter mitosis before completion of DNA replication (8-11). In Saccharomyces cerevisiae, Cdc6, the probable homolog of Schizosaccharomyces pombe Cdc18, was reported to have a checkpoint function, acting positively at the initiation of DNA replication and negatively at the entry to mitosis (12). However, homologs of fission yeast Cut5 and Cdtl had not been found in Saccharomyces cerevisiae.Though several checkpoint mutations and the corresponding genes have been isolated, the mechanism sensing the actual DNA damage or replication block remains obscure. Recently, Navas et al. (13) reported that Pol2, the catalytic subunit of Saccharomyces cerevisiae DNA polymerase II, is important for the S-phase checkpoint and they proposed that DNA polymerase II acts as a sensor of DNA replication. In Saccharomyces cerevisiae, DNA polymerase II(e) and two other DNA polymerases, I(a) and III(8), are essential for chromosomalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.DNA replication (for review, se...

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Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

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Here, we present evidence that fortilin and MCL1 are capable of functioning as antiapoptotic proteins independently of each other. Using a robust small interfering RNA (siRNA)-mediated gene silencing system developed in our laboratory, we analyzed the cytoprotective effects of fortilin and MCL1 together and apart in U2OS cell lines exposed to 5-fluorouracil (5-FU) in both monoclonal and polyclonal cell populations. When MCL1 was silenced by MCL1-targeted siRNA, fortilin was still able to protect cells from 5-FU-induced cytotoxicity in a dosedependent manner. Conversely, when fortilin was silenced by fortilin-targeted siRNA, MCL1 was also able to protect cells from 5-FU-induced cytotoxicity in a dosedependent manner. Together, these data clearly suggest that fortilin and MCL1 can exert their cytoprotective activities independently of each other. The silencing of fortilin and MCL1 did not qualitatively change the subcellular localization of MCL1 and fortilin, respectively. The biological significance of fortilin-MCL1 interaction may be that it increases cellular resistance to apoptosis by allowing MCL1, an independently antiapoptotic protein, to stabilize another independently antiapoptotic protein, fortilin.Fortilin is a 172-amino acid polypeptide that was originally identified by yeast two-hybrid library screening as a molecule that specifically interacted with MCL1, a protein of the antiapoptotic Bcl-2 family (1). Fortilin is also known as translationally controlled tumor protein (2, 3). Early analyses of fortilin in our laboratory revealed that its amino acid sequence is highly evolutionarily conserved; that fortilin is ubiquitous in normal tissues, especially in liver and kidney; and that it localizes in both the nucleus and cytosol. In addition, we found that its overexpression prevents HeLa and U2OS cells from undergoing etoposide-induced apoptosis and that antisense depletion of fortilin can induce MCF-7 cells to die spontaneously. Taken together, these findings have established fortilin as a unique antiapoptotic protein.Because the amino acid sequence of fortilin does not resemble that of either Bcl-2 family proteins or IAPs (inhibitor of apoptosis proteins) and because fortilin specifically interacts with MCL1, an antiapoptotic Bcl-2 family protein, we first hypothesized that the antiapoptotic function of fortilin is mediated through MCL1. Intriguingly, we found that fortilin interacted only with MCL1, not with other Bcl-2 family proteins, suggesting that fortilin might be an MCL1-specific cofactor in the regulation of apoptosis (4). At that time, we devised our first-generation small interfering RNA (siRNA) 1 system, in which we could specifically and effectively knock down MCL1 or fortilin expression in vivo. Using this system, we unexpectedly found that MCL1 depletion by siRNA targeting MCL1 (siRNA MCL1 ) drastically reduced the intracellular concentration of fortilin, whereas the siRNA targeting fortilin (siR-NA Fortilin )-mediated depletion of fortilin did not affect the intracellular concentration ...