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Joel A Huberman

Publications and source records attributed to Joel A Huberman.

12 recordsLinked to original sources
PubMed2006

Production of reactive oxygen species in response to replication stress and inappropriate mitosis in fission yeast.

Previous studies have indicated that replication stress can trigger apoptosis-like cell death, accompanied (where tested) by production of reactive oxygen species (ROS), in mammalian cells and budding yeast (Saccharomyces cerevisiae). In mammalian cells, inappropriate entry into mitosis also leads to cell death. Here, we report similar responses in fission yeast (Schizosaccharomyces pombe). We used ROS- and death-specific fluorescent stains to measure the effects of mutations in replication initiation and checkpoint genes in fission yeast on the frequencies of ROS production and cell death. We found that certain mutant alleles of each of the four tested replication initiation genes caused elevated ROS and cell death. Where tested, these effects were not enhanced by checkpoint-gene mutations. Instead, when cells competent for replication but defective in both the replication and damage checkpoints were treated with hydroxyurea, which slows replication fork movement, the frequencies of ROS production and cell death were greatly increased. This was a consequence of elevated CDK activity, which permitted inappropriate entry into mitosis. Thus, studies in fission yeast are likely to prove helpful in understanding the pathways that lead from replication stress and inappropriate mitosis to cell death in mammalian cells.

Cell Cycle Proteins↗
PubMed2005

Apoptosis in budding yeast caused by defects in initiation of DNA replication.

Apoptosis in metazoans is often accompanied by the destruction of DNA replication initiation proteins, inactivation of checkpoints and activation of cyclin-dependent kinases, which are inhibited by checkpoints that directly or indirectly require initiation proteins. Here we show that, in the budding yeast Saccharomyces cerevisiae, mutations in initiation proteins that attenuate both the initiation of DNA replication and checkpoints also induce features of apoptosis similar to those observed in metazoans. The apoptosis-like phenotype of initiation mutants includes the production of reactive oxygen species (ROS) and activation of the budding-yeast metacaspase Yca1p. In contrast to a recent report that activation of Yca1p only occurs in lysed cells and does not contribute to cell death, we found that, in at least one initiation mutant, Yca1p activation occurs at an early stage of cell death (before cell lysis) and contributes to the lethal effects of the mutation harbored by this strain. Apoptosis in initiation mutants is probably caused by DNA damage associated with the combined effects of insufficient DNA replication forks to completely replicate the genome and defective checkpoints that depend on initiation proteins and/or replication forks to restrain subsequent cell-cycle events until DNA replication is complete. A similar mechanism might underlie the proapoptotic effects associated with the destruction of initiation and checkpoint proteins during apoptosis in mammals, as well as genome instability in initiation mutants of budding yeast.

Apoptosis↗
PubMed2005

Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage.

The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.

Alkylation↗
PubMed2004

On the slowing of S phase in response to DNA damage in fission yeast.

Eukaryotic cells slow their progression through S phase upon DNA damage. The mechanism that leads to this slowing is called the intra-S-phase checkpoint. Previous studies demonstrated that in the fission yeast Schizosaccharomyces pombe this checkpoint is mediated by a pathway that includes Rad3 (similar to human ATR and ATM) and Cds1 (similar to human Chk1 and Chk2). Here we present evidence that a major downstream target of this pathway is the cyclin-dependent kinase, Cdc2. We also present evidence suggesting that the intra-S-phase checkpoint makes a relatively minor contribution to the survival of cells with damaged DNA.

CDC2 Protein Kinase↗
PubMed2004

Enforcement of late replication origin firing by clusters of short G-rich DNA sequences.

Previous studies in budding yeast suggested that the default firing time of most DNA replication origins is early in S phase and that origins can be forced to fire later by proximity to certain cis-acting sequences. However, these cis-acting sequences were not well defined. We have attempted to characterize cis-acting sequences that affect replication timing in the fission yeast. We identified a stretch of 200 bp that was sufficient to compel nearby origins to fire late. The 200-bp stretch was able to force an origin to fire late whether adjacent to the origin or approximately 800 bp away in opposite orientation. The stretch contains a cluster of three close matches to a G-rich, 10-bp late consensus sequence (LCS). The three LCS elements cooperate with each other and with other sequences within the 200-bp stretch to enforce late replication. Although only a few origins that fire in very late S phase have been identified in fission yeast, all of them are located close to a cluster of LCS elements.

Base Sequence↗
PubMed2003

Apoptosis-like yeast cell death in response to DNA damage and replication defects.

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.

Alkylating Agents↗
PubMed2003

Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone.

Previous data from our laboratory suggested that replication of mammalian mitochondrial DNA initiates exclusively at or near to the formerly designated origin of heavy strand replication, OH, and proceeds unidirectionally from that locus. New results obtained using two-dimensional agarose gel electrophoresis of replication intermediates demonstrate that replication of mitochondrial DNA initiates from multiple origins across a broad zone. After fork arrest near OH, replication is restricted to one direction only. The initiation zone of bidirectional replication includes the genes for cytochrome b and NADH dehydrogenase subunits 5 and 6.

Animals↗
PubMed2003

Early-replicating heterochromatin.

Euchromatin, which has an open structure and is frequently transcribed, tends to replicate in early S phase. Heterochromatin, which is more condensed and rarely transcribed, usually replicates in late S phase. Here, we report significant deviation from this correlation in the fission yeast, Schizosaccharomyces pombe. We found that heterochromatic centromeres and silent mating-type cassettes replicate in early S phase. Only heterochromatic telomeres replicate in late S phase. Research in other laboratories has shown that occasionally other organisms also replicate some of their heterochromatin in early S phase. Thus, late replication is not an obligatory feature of heterochromatin.

Centromere↗
PubMed2003

Motifs in Schizosaccharomyces pombe ars3002 important for replication origin activity in Saccharomyces cerevisiae.

Ars3002 is an efficient single-copy replication origin in the fission yeast, Schizosaccharomyces pombe. In a previous study, we tested the effects of consecutive approximately 50-bp deletions throughout ars3002 on the replication efficiency of those origins in S. pombe. Here we report the results of our use of the same approximately 50-bp deletions to test the hypothesis that some of the cis-acting sequences important for replication origin activity in fission yeast might be conserved in the evolutionarily distant budding yeast, Saccharomyces cerevisiae. We found that in most cases there was no correlation between the effects of particular mutations in S. pombe and in S. cerevisiae. We conclude that it is unlikely that any of the cis-acting sequences recognised by homologous replication proteins is conserved between these two yeast species.

Base Sequence↗
PubMed2002

A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway.

The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood. Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast, Schizosaccharomyces pombe, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways. The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase epsilon) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition). These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.

Cell Cycle Proteins↗