Bidyut K. Mohanty

Binding of the Replication Terminator Protein Fob1p to the Ter Sites of Yeast Causes Polar Fork Arrest

Fob1p protein has been implicated in the termination of replication forks at the two tandem termini present in the non-transcribed spacer region located between the sequences encoding the 35 S and the 5 S RNAs of Saccharomyces cerevisiae. However, the biochemistry and mode of action of this protein were previously unknown. We have purified the Fob1p protein to near-homogeneity, and we developed a novel technique to show that it binds specifically to the Ter1 and Ter2 sequences. Interestingly, the two sequences share no detectable homology. We present two lines of evidence showing that the interaction of the Fob1p with the Ter sites causes replication termination. First, a mutant of FOB1, L104S that significantly reduced the binding of the mutant form of the protein to the tandem Ter sites, also failed to promote replication termination in vivo. The mutant did not diminish nucleolar transport, and interaction of the mutant form of Fob1p with itself and with another protein encoded in the locus YDR026C suggested that the mutation did not cause global misfolding of the protein. Second, DNA site mutations in the Ter sequences that separately and specifically abolished replication fork arrest at Ter1 or Ter2 also eliminated sequence-specific binding of the Fob1p to the two sites. The work presented here definitively established Ter DNA-Fob1p interaction as an important step in fork arrest.

Hydroxyurea Sensitivity Reveals a Role for ISC1 in the Regulation of G2/M

Saccharomyces cerevisiae cells lacking ISC1 (inositol phosphosphingolipase C) exhibit sensitivity to genotoxic agents such as methyl methanesulfonate and hydroxyurea (HU). Cell cycle analysis by flow cytometry revealed a G2/M block in isc1Δ cells when treated with methyl methanesulfonate or HU. Further investigation revealed that the levels of Cdc28 phosphorylated on Tyr-19, which plays an essential role in the regulation of the G2/M checkpoint, were higher in synchronized and asynchronous cells lacking ISC1 in response to HU. Use of a Cdc28-Y19F mutant protected isc1Δ from the G2/M block. In wild type cells, HU induced a loss of the Swe1p kinase, the enzyme that phosphorylates Cdc28-Tyr-19, correlating with resumption of the cell cycle. In the isc1Δ cells, however, the levels of Swe1p remained at sustained high levels in response to HU. Significantly, deletion of SWE1 in an isc1Δ background overcame the G2/M block in response to HU. The double isc1Δ/swe1Δ mutant also overcame the growth defect on HU. Taken together, these findings implicate Isc1p as an upstream regulator of Swe1p levels and stability and Cdc28-Tyr-19 phosphorylation, in effect signaling recovery from the effects of genotoxic stress and allowing G2/M progression.

Identification of C18:1-Phytoceramide as the Candidate Lipid Mediator for Hydroxyurea Resistance in Yeast

Background: Deletion of the sphingolipid enzyme Isc1 in yeast makes cells sensitive to hydroxyurea. Results: Phytoceramides are the main sphingolipid involved in yeast cell response to hydroxyurea toxicity. Conclusion: C18:1-phytoceramides are identified as the specific ceramide that provide protection from hydroxyurea in a Cdc55-dependent manner. Significance: This is the first specific sphingolipid species identified to play a role in the genotoxic response pathway. Recent studies showed that deletion of ISC1, the yeast homologue of the mammalian neutral sphingomyelinase, resulted in an increased sensitivity to hydroxyurea (HU). This raised an intriguing question as to whether sphingolipids are involved in pathways initiated by HU. In this study, we show that HU treatment led to a significant increase in Isc1 activity. Analysis of sphingolipid deletion mutants and pharmacological analysis pointed to a role for ceramide in mediating HU resistance. Lipid analysis revealed that HU induced increases in phytoceramides in WT cells but not in isc1Δ cells. To probe functions of specific ceramides, we developed an approach to supplement the medium with fatty acids. Oleate (C18:1) was the only fatty acid protecting isc1Δ cells from HU toxicity in a ceramide-dependent manner. Because phytoceramide activates protein phosphatases in yeast, we evaluated the role of CDC55, the regulatory subunit of ceramide-activated protein phosphatase PP2A. Overexpression of CDC55 overcame the sensitivity to HU in isc1Δ cells. However, addition of oleate did not protect the isc1Δ,cdc55Δ double mutant from HU toxicity. These results demonstrate that HU launches a lipid pathway mediated by a specific sphingolipid, C18:1-phytoceramide, produced by Isc1, which provides protection from HU by modulating Swe1 levels through the PP2A subunit Cdc55.

Replication Fork Arrest and rDNA Silencing Are Two Independent and Separable Functions of the Replication Terminator Protein Fob1 of Saccharomyces cerevisiae

The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other as well as functionally separable. First, a Fob1 ortholog of Saccharomyces bayanus expressed in a fob1Δ strain of S. cerevisiae restored polar fork arrest at Ter but not rDNA silencing. Second, a mutant form (I407T) of S. cerevisiae Fob1 retained normal fork arresting activity but was partially defective in rDNA silencing. We further show that the silencing defect of S. bayanus Fob1 and the Ι407Τ mutant of S. cerevisiae Fob1 were caused by the failure of the proteins to interact with two members of the S. cerevisiae RENT complex, namely S. cerevisiae Sir2 and S. cerevisiae Net1. Third, deletions of the intra-S phase checkpoint proteins Tof1 and Csm3 abolished fork arrest by Fob1 at Ter without causing loss of silencing. Taken together, the data support the conclusion that unlike some other functions of Fob1, rDNA silencing at Ter is independent of fork arrest.

Contrasting Roles of Checkpoint Proteins as Recombination Modulators at Fob1-Ter Complexes with or without Fork Arrest

ABSTRACT The replication terminator protein Fob1 of Saccharomyces cerevisiae specifically interacts with two tandem Ter sites (replication fork barriers) located in the nontranscribed spacer of ribosomal DNA (rDNA) to cause polar fork arrest. The Fob1-Ter complex is multifunctional and controls other DNA transactions such as recombination by multiple mechanisms. Here, we report on the regulatory roles of the checkpoint proteins in the initiation and progression of recombination at Fob1-Ter complexes. The checkpoint adapter proteins Tof1 and Csm3 either positively or negatively controlled recombination depending on whether it was provoked by polar fork arrest or by transcription, respectively. The absolute requirements for these proteins for inducing recombination at an active replication terminus most likely masked their negative modulatory role at a later step of the process. Other checkpoint proteins of the checkpoint adapter/mediator class such as Mrc1 and Rad9, which channel signals from the sensor to the effector kinase, tended to suppress recombination at Fob1-Ter complexes regardless of how it was initiated. We have also discovered that the checkpoint sensor kinase Mec1 and the effector Rad53 were positive modulators of recombination initiated by transcription but had little effect on recombination at Ter. The work also showed that the two pathways were Rad52 dependent but Rad51 independent. Since Ter sites occur in the intergenic spacer of rDNA from yeast to humans, the mechanism is likely to be of widespread occurrence.

Cellular Morphogenesis Under Stress Is Influenced by the Sphingolipid Pathway Gene ISC1 and DNA Integrity Checkpoint Genes in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, replication stress induced by hydroxyurea (HU) and methyl methanesulfonate (MMS) activates DNA integrity checkpoints; in checkpoint-defective yeast strains, HU treatment also induces morphological aberrations. We find that the sphingolipid pathway gene ISC1, the product of which catalyzes the generation of bioactive ceramides from complex sphingolipids, plays a novel role in determining cellular morphology following HU/MMS treatment. HU-treated isc1Δ cells display morphological aberrations, cell-wall defects, and defects in actin depolymerization. Swe1, a morphogenesis checkpoint regulator, and the cell cycle regulator Cdk1 play key roles in these morphological defects of isc1Δ cells. A genetic approach reveals that ISC1 interacts with other checkpoint proteins to control cell morphology. That is, yeast carrying deletions of both ISC1 and a replication checkpoint mediator gene including MRC1, TOF1, or CSM3 display basal morphological defects, which increase following HU treatment. Interestingly, strains with deletions of both ISC1 and the DNA damage checkpoint mediator gene RAD9 display reduced morphological aberrations irrespective of HU treatment, suggesting a role for RAD9 in determining the morphology of isc1Δ cells. Mechanistically, the checkpoint regulator Rad53 partially influences isc1Δ cell morphology in a dosage-dependent manner.

The Intra-S Phase Checkpoint Protein Tof1 Collaborates with the Helicase Rrm3 and the F-box Protein Dia2 to Maintain Genome Stability in Saccharomyces cerevisiae

The intra-S phase checkpoint protein complex Tof1/Csm3 of Saccharomyces cerevisiae antagonizes Rrm3 helicase to modulate replication fork arrest not only at the replication termini of rDNA but also at strong nonhistone protein binding sites throughout the genome. We investigated whether these checkpoint proteins acted either antagonistically or synergistically with Rrm3 in mediating other important functions such as maintenance of genome stability. High retromobility of a normally quiescent retrovirus-like transposable element Ty1 of S. cerevisiae is a form of genome instability, because the transposition events induce mutations. We measured the transposition of Ty1 in various genetic backgrounds and discovered that Tof1 suppressed excessive retromobility in collaboration with either Rrm3 or the F-box protein Dia2. Although both Rrm3 and Dia2 are believed to facilitate fork movement, fork stalling at DNA-protein complexes did not appear to be a major contributor to enhancement of retromobility. Absence of the aforementioned proteins either individually or in pair-wise combinations caused karyotype changes as revealed by the altered migrations of the individual chromosomes in pulsed field gels. The mobility changes were RNase H-resistant and therefore, unlikely to have been caused by extensive R loop formation. These mutations also resulted in alterations of telomere lengths. However, the latter changes could not fully account for the magnitude of the observed karyotypic alterations. We conclude that unlike other checkpoint proteins that are known to be required for elevated retromobility, Tof1 suppressed high frequency retrotransposition and maintained karyotype stability in collaboration with the aforementioned proteins.

8 Termination of DNA Replication

Sequence-specific replication termini (Ter) occur at the antipodes, i.e., 180° from the replication origins of circular bacterial chromosomes (and in some plasmid replicons, e.g., R6K), and ensure that termination occurs within a defined region. Meeting of the two forks within this region is necessary for efficient resolution of oligomers formed by an odd number of crossovers between the two daughter chromosomes, and dimer resolution is essential for proper chromosome segregation (see details in the next section). In contrast, although most eukaryotic replicons do not appear to have sequence-specific replication termini, these are present at specific regions of the chromosome that require polarized replication fork movement necessary for carrying out specific physiological functions; e.g., mating-type switching in Schizosaccharomyces pombe . This review covers the mechanism of replication fork arrest at the natural replication termini and pause sites of prokaryotes and eukaryotes and explores their possible regulatory roles in the control of replication fork movement through these regions and in other DNA transactions. Only the newer information on replication termination and fork pausing that has been uncovered since the publication of an earlier review (Bastia and Mohanty 1996) is discussed in this chapter. REPLICATION TERMINATION IN PROKARYOTES Most circular prokaryotic chromosomes terminate replication at sequence-specific termini called Ter sites. Such sites (also called Replication Fork Barriers or RFB, Replication Termination Sites or RTS, etc.) are also present at specific regions of the eukaryotic chromosomes; e.g., at the nontranscribed spacers of rDNA of most eukaryotes from yeast to man (Bastia and Mohanty 1996; Rothstein...

Finding pathway-modulating genes from a novel Ontology Fingerprint-derived gene network

To enhance our knowledge regarding biological pathway regulation, we took an integrated approach, using the biomedical literature, ontologies, network analyses and experimental investigation to infer novel genes that could modulate biological pathways. We first constructed a novel gene network via a pairwise comparison of all yeast genes’ Ontology Fingerprints—a set of Gene Ontology terms overrepresented in the PubMed abstracts linked to a gene along with those terms’ corresponding enrichment P-values. The network was further refined using a Bayesian hierarchical model to identify novel genes that could potentially influence the pathway activities. We applied this method to the sphingolipid pathway in yeast and found that many top-ranked genes indeed displayed altered sphingolipid pathway functions, initially measured by their sensitivity to myriocin, an inhibitor of de novo sphingolipid biosynthesis. Further experiments confirmed the modulation of the sphingolipid pathway by one of these genes, PFA4, encoding a palmitoyl transferase. Comparative analysis showed that few of these novel genes could be discovered by other existing methods. Our novel gene network provides a unique and comprehensive resource to study pathway modulations and systems biology in general.

Identification and characterization of an hnRNP E1 translational silencing motif

Non-canonical transforming growth factor β (TGFβ) signaling through protein kinase B (Akt2) induces phosphorylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) at serine-43 (p-hnRNP E1). This post-translational modification (PTM) of hnRNP E1 promotes its dissociation from a 3′ untranslated region (UTR) nucleic acid regulatory motif, driving epithelial to mesenchymal transition (EMT) and metastasis. We have identified an hnRNP E1 consensus-binding motif and genomically resolved a subset of genes in which it is contained. This study characterizes the binding kinetics of the consensus-binding motif and hnRNP E1, its various K-homology (KH) domains and p-hnRNP E1. Levels of p-hnRNP E1 are highly upregulated in metastatic cancer cells and low in normal epithelial tissue. We show a correlation between this PTM and levels of Akt2 and its activated form, phosphorylated serine-474 (p-Akt2). Using cellular progression models of metastasis, we observed a signature high level of Akt2, p-Akt2 and p-hnRNP E1 protein expression, coupled to a significantly reduced level of total hnRNP E1 in metastatic cells. Genes that are translationally silenced by hnRNP E1 and expressed by its dissociation are highly implicated in the progression of EMT and metastasis. This study provides insight into a non-canonical TGFβ signaling cascade that is responsible for inducing EMT by aberrant expression of hnRNP E1 silenced targets. The relevance of this system in metastatic progression is clearly shown in cellular models by the high abundance of p-hnRNP E1 and low levels of hnRNP E1. New insights provided by the resolution of this molecular mechanism provide targets for therapeutic intervention and give further insight into the role of the TGFβ microenvironment.