scholarly journals Faculty Opinions recommendation of Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation.

2011 ◽  
Author(s):  
Joel Huberman
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Essential Role ◽  
Replication Initiation ◽  
Dna Replication Initiation

scholarly journals Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation

2011 ◽  
Vol 195 (3) ◽  
pp. 387-401 ◽  
Author(s):  
Seiji Matsumoto ◽  
Motoshi Hayano ◽  
Yutaka Kanoh ◽  
Hisao Masai
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Fork ◽  
Essential Role ◽  
Replication Initiation ◽  
Origin Firing ◽  
Physiological Conditions ◽  
Dna Replication Initiation ◽  
Initiation Of Dna Replication

Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication that potentially regulates timing and locations of replication origin firing. Here, we show that viability of fission yeast hsk1Δ cells can be restored by loss of mrc1, which is required for maintenance of replication fork integrity, by cds1Δ, or by a checkpoint-deficient mutant of mrc1. In these mutants, normally inactive origins are activated in the presence of hydroxyurea and binding of Cdc45 to MCM is stimulated. mrc1Δ bypasses hsk1Δ more efficiently because of its checkpoint-independent inhibitory functions. Unexpectedly, hsk1Δ is viable at 37°C. More DNA is synthesized, and some dormant origins fire in the presence of hydroxyurea at 37°C. Furthermore, hsk1Δ bypass strains grow poorly at 25°C compared with higher temperatures. Our results show that Hsk1 functions for DNA replication can be bypassed by different genetic backgrounds as well as under varied physiological conditions, providing additional evidence for plasticity of the replication program in eukaryotes.

Essential role of human CDT1 in DNA replication and chromatin licensing

2002 ◽  
Vol 115 (7) ◽  
pp. 1435-1440 ◽  
Author(s):  
Mickael Rialland ◽  
Francesco Sola ◽  
Corrado Santocanale
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Complex Formation ◽  
Fission Yeast ◽  
Origin Recognition Complex ◽  
Essential Role ◽  
Large Body ◽  
Transformed Cells ◽  
Minichromosome Maintenance

Formation of pre-replicative complexes at origins is an early cell cycle event essential for DNA duplication. A large body of evidence supports the notion that Cdc6 protein, through its interaction with the origin recognition complex, is required for pre-replicative complex assembly by loading minichromosome maintenance proteins onto DNA. In fission yeast and Xenopus, this reaction known as the licensing of chromatin for DNA replication also requires the newly identified Cdt1 protein. We studied the role of hCdt1 protein in the duplication of the human genome by antibody microinjection experiments and analyzed its expression during the cell cycle in human non-transformed cells. We show that hCdt1 is essential for DNA replication in intact human cells, that it executes its function in a window of the cell cycle overlapping with pre-replicative complex formation and that it is necessary for the loading of minichromosome maintenance proteins onto chromatin. Intriguingly, we observed that hCdt1 protein, in contrast to other licensing factors, is already present in serum-deprived G0 arrested cells and its levels increase only marginally upon re-entry in the cell cycle.

scholarly journals Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae

2020 ◽  
Vol 10 (6) ◽  
pp. 2057-2068 ◽  
Author(s):  
Jessica R. Eisenstatt ◽  
Lars Boeckmann ◽  
Wei-Chun Au ◽  
Valerie Garcia ◽  
Levi Bursch ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Initiation ◽  
Centromeric Chromatin ◽  
Dna Replication Initiation ◽  
Link Type ◽  
Genome Wide ◽  
A Genome ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.

Fission yeast Cdc23 interactions with DNA replication initiation proteins

2002 ◽  
Vol 41 (5) ◽  
pp. 342-348 ◽  
Author(s):  
Elizabeth Hart ◽  
John Bryant ◽  
Karen Moore ◽  
Stephen Aves
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Dna Replication Initiation ◽  
Replication Initiation Proteins

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria

2019 ◽  
Vol 16 (3) ◽  
pp. 272-277 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Anders Løbner-Olesen
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
Cyclic Peptide ◽  
Replication Initiation ◽  
False Positive Result ◽  
Over Expression ◽  
Initiator Protein ◽  
Dna Replication Initiation ◽  
Cellular Processes ◽  
Initiation Of Dna Replication

Background:One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.Materials and Methods:Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.Results:The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.Conclusion:We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

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