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J Leatherwood

Publications and source records attributed to J Leatherwood.

13 recordsLinked to original sources
PubMed2001

Control of DNA rereplication via Cdc2 phosphorylation sites in the origin recognition complex.

Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G(2) and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

Amino Acid Sequence↗
PubMed2000

Functions of fission yeast orp2 in DNA replication and checkpoint control.

orp2 is an essential gene of the fission yeast Schizosaccharomyces pombe with 22% identity to budding yeast ORC2. We isolated temperature-sensitive alleles of orp2 using a novel plasmid shuffle based on selection against thymidine kinase. Cells bearing the temperature-sensitive allele orp2-2 fail to complete DNA replication at a restrictive temperature and undergo cell cycle arrest. Cell cycle arrest depends on the checkpoint genes rad1 and rad3. Even when checkpoint functions are wild type, the orp2-2 mutation causes high rates of chromosome and plasmid loss. These phenotypes support the idea that Orp2 is a replication initiation factor. Selective spore germination allowed analysis of orp2 deletion mutants. These experiments showed that in the absence of orp2 function, cells proceed into mitosis despite a lack of DNA replication. This suggests either that the Orp2 protein is a part of the checkpoint machinery or more likely that DNA replication initiation is required to induce the replication checkpoint signal.

Base Sequence↗
PubMed2000

Where does DNA replication start in archaea?

Genome-wide measures of DNA strand composition have been used to find archaeal DNA replication origins. Archaea seem to replicate using a single origin (as do eubacteria) even though archaeal replication factors are more like those of eukaryotes.

Archaea↗
PubMed1998

Emerging mechanisms of eukaryotic DNA replication initiation.

Recent research has focused on proteins important for early steps in replication in eukaryotes, and particularly on Cdc6/Cdc18, the MCMs, and Cdc45. Although it is still unclear exactly what role these proteins play, it is possible that they are analogous to initiation proteins in prokaryotes. One specific model is that MCMs form a hexameric helicase at replication forks, and Cdc6/Cdc18 acts as a 'clamp-loader' required to lock the MCMs around DNA. The MCMs appear to be the target of Cdc7-Dbf4 kinase acting at individual replication origins. Finally, Cdc45 interacts with MCMs and may shed light on how cyclin-dependent kinases activate DNA replication.

Animals↗
PubMed1998

Negative regulation of Cdc18 DNA replication protein by Cdc2.

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

CDC2 Protein Kinase↗
PubMed1997

Pch1(+), a second essential C-type cyclin gene in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe gene pch1(+) (pombe cyclin C homology) was isolated in a two-hybrid screen for proteins that interact with Cdc2. The cyclin box region of Pch1 protein shares greatest sequence identity with mammalian and Drosophila C-type cyclins ( approximately 33% identity). Pch1 is significantly less similar to Mcs2 (19% identity), a second member of the C-type cyclin family in S. pombe. Cdc2 co-precipitates with Pch1 in S. pombe cell lysates, although Cdc2 may not be the major catalytic partner of a Pch1 kinase in vivo. Purified Pch1-associated kinase phosphorylated myelin basic protein, histone H1, and a peptide corresponding to the carboxyl-terminal domain repeat of RNA polymerase II. The amount of pch1 mRNA does not oscillate during the cell cycle, as is the case for mRNA transcripts of other C-type cyclin genes. Deltapch1 cells are inviable, therefore S. pombe has two essential genes that encode members of the C-type cyclin family, pch1(+) and mcs2(+). The Deltapch1 mutation causes pleiotropic morphological defects and an associated growth deficiency, but loss of Pch1 activity does not result in a cdc cell cycle-arrest phenotype.

Alleles↗
PubMed1996

Interaction of Cdc2 and Cdc18 with a fission yeast ORC2-like protein.

In fission yeast, Cdc2 kinase has both positive and negative roles in regulating DNA replication, being first necessary for the transition from G1 to S phase and later required to prevent the re-initiation of DNA replication during G2. We report here that Cdc2 interacts with Orp2, a protein similar to the Orc2 replication factor subunit of Saccharomyces cerevisiae origin recognition complex (ORC). ORC binds chromosomal origins and is essential for chromosomal replication initiation. Fission yeast Orp2 is required for DNA replication and interacts with the rate-limiting replication activator Cdc18. Cells lacking Orp2 undergo aberrant mitosis, indicating that Orp2 is involved in generating a checkpoint signal. These findings suggest that ORC functions are conserved among eukaryotes and provide evidence that Cdc2 controls DNA replication initiation by acting directly at chromosomal origins.

Amino Acid Sequence↗
PubMed1990

A potent GAL4 derivative activates transcription at a distance in vitro.

Transcription of a typical eukaryotic gene by RNA polymerase II is activated by proteins bound to sites found near the beginning of the gene as well as to sites, called enhancers, located a great distance from the gene. According to one view, the primary difference between an activator that can work at a large distance and one that cannot is that the former bears a particularly strong activating region; the stronger the activating region, the more readily the activator interacts with its target bound near the transcriptional start site, with the intervening DNA looping out to accommodate the reaction. One alternative view is that the effect of proteins bound to enhancers might require some special aspect of cellular or chromosome structure. Consistent with the first view, an activator bearing an unusually potent activating region can stimulate transcription of a mammalian gene in a HeLa nuclear extract when bound as far as 1.3 kilobase pairs upstream or 320 base pairs downstream of the transcriptional start site.

Activating Transcription Factors↗
PubMed1989

An amino-terminal fragment of GAL4 binds DNA as a dimer.

GAL4 is a yeast transcriptional activator protein that binds to specific 2-fold rotationally symmetric sites on DNA and stimulates transcription of the genes required for galactose catabolism. The DNA binding region of the protein is located within the first 74 amino acids and contains a "zinc finger" sequence motif. We show that a polypeptide comprising the first 147 amino acids of GAL4, designated GAL4 (1-147), binds DNA as a dimer in vitro. Although a protein containing only the first 74 amino acids, designated GAL4 (1-74), binds DNA specifically, its affinity is reduced relative to GAL4 (1-147). Addition of the strong dimerization domain of lambda repressor to GAL4 (1-74) generates a protein that binds as tightly as GAL4 (1-147). GAL4 (1-147) makes rotationally symmetric contacts with its recognition site when assayed by DNase I, exonuclease III and hydroxyl radical footprinting and by phosphate ethylation interference. Binding of GAL4 (1-147) in vitro requires either zinc or cadmium.

Amino Acids↗
PubMed1989

Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

Base Sequence↗