scholarly journals Sld2, Which Interacts with Dpb11 inSaccharomyces cerevisiae, Is Required for Chromosomal DNA Replication

1998 ◽  
Vol 18 (10) ◽  
pp. 6102-6109 ◽  
Author(s):  
Yoichiro Kamimura ◽  
Hiroshi Masumoto ◽  
Akio Sugino ◽  
Hiroyuki Araki
Keyword(s):  
Dna Replication ◽  
Temperature Shift ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Chromosomal Dna ◽  
Autonomously Replicating Sequence ◽  
Complementation Groups ◽  
Synthetically Lethal ◽  
Dna Polymerase Ii

ABSTRACT The DPB11 gene, which genetically interacts with DNA polymerase II (ɛ), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint inSaccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal withdpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11suppressed the temperature-sensitive growth defect caused bysld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25°C but not at 34°C when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25°C. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.

scholarly journals MEIOTIC RECOMBINATION AND DNA SYNTHESIS IN A NEW CELL CYCLE MUTANT OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1978 ◽  
Vol 90 (1) ◽  
pp. 49-68
Author(s):  
Yona Kassir ◽  
Giora Simchen
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Division ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Essential Function ◽  
Normal Meiosis

ABSTRACT Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.

scholarly journals A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function.

1997 ◽  
Vol 17 (4) ◽  
pp. 2136-2142 ◽  
Author(s):  
M E Budd ◽  
J L Campbell
Keyword(s):  
Dna Replication ◽  
Genetic Interactions ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Physical Interaction ◽  
Replicative Helicase ◽  
Direct Physical Interaction ◽  
Dna Replication And Repair ◽  
Essential Function ◽  
Synthetically Lethal

We have recently described a new helicase, the Dna2 helicase, that is essential for yeast DNA replication. We now show that the yeast FEN-1 (yFEN-1) nuclease interacts genetically and biochemically with Dna2 helicase. FEN-1 is implicated in DNA replication and repair in yeast, and the mammalian homolog of yFEN-1 (DNase IV, FEN-1, or MF1) participates in Okazaki fragment maturation. Overproduction of yFEN-1, encoded by RAD27/RTH1, suppresses the temperature-sensitive growth of dna2-1 mutants. Overproduction of Dna2 suppresses the rad27/rth1 delta temperature-sensitive growth defect. dna2-1 rad27/rth1 delta double mutants are inviable, indicating that the mutations are synthetically lethal. The genetic interactions are likely due to direct physical interaction between the two proteins, since both epitope-tagged yFEN-1 and endogenous yFEN-1 coimmunopurify with tagged Dna2. The simplest interpretation of these data is that one of the roles of Dna2 helicase is associated with processing of Okazaki fragments.

The osmotic integrity of the yeast cell requires a functional PKC1 gene product

1992 ◽  
Vol 12 (11) ◽  
pp. 4896-4905
Author(s):  
G Paravicini ◽  
M Cooper ◽  
L Friedli ◽  
D J Smith ◽  
J L Carpentier ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Yeast Genome ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Electron Microscopic ◽  
Gene Encoding ◽  
Mutant Strains ◽  
Complementation Groups ◽  
Mammalian Protein

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.

scholarly journals The osmotic integrity of the yeast cell requires a functional PKC1 gene product.

1992 ◽  
Vol 12 (11) ◽  
pp. 4896-4905 ◽  
Author(s):  
G Paravicini ◽  
M Cooper ◽  
L Friedli ◽  
D J Smith ◽  
J L Carpentier ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Yeast Genome ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Electron Microscopic ◽  
Gene Encoding ◽  
Mutant Strains ◽  
Complementation Groups ◽  
Mammalian Protein

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.

scholarly journals Suppressors of a Saccharomyces cerevisiae pkc1 mutation identify alleles of the phosphatase gene PTC1 and of a novel gene encoding a putative basic leucine zipper protein.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1275-1285 ◽  
Author(s):  
K N Huang ◽  
L S Symington
Keyword(s):  
Protein Kinase ◽  
Leucine Zipper ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Basic Leucine Zipper ◽  
Mapk Cascades ◽  
Synthetically Lethal

Abstract The PKC1 gene product, protein kinase C, regulates a mitogen-activated protein kinase (MAPK) cascade, which is implicated in cell wall metabolism. Previously, we identified the pkc1-4 allele in a screen for mutants with increased rates of recombination, indicating that PKC1 may also regulate DNA metabolism. The pkc1-4 allele also conferred a temperature-sensitive (ts) growth defect. Extragenic suppressors were isolated that suppress both the ts and hyperrecombination phenotypes conferred by the pkc1-4 mutation. Eight of these suppressors for into two complementation groups, designated KCS1 and KCS2. KCS1 was cloned and found to encode a novel protein with homology to the basic leucine zipper family of transcription factors. KCS2 is allelic with PTC1, a previously identified type 2C serine/threonine protein phosphatase. Although mutation of either KCS1 or PTC1 causes little apparent phenotype, the kcs1 delta ptc1 delta double mutant fails to grow at 30 degrees. Furthermore, the ptc1 deletion mutation is synthetically lethal in combination with a mutation in MPK1, which encodes a MAPK homologue proposed to act in the PKC1 pathway. Because PTC1 was initially isolated as a component of the Hog1p MAPK pathway, it appears that these two MAPK cascades share a common regulatory feature.

scholarly journals THE SELECTION OF S. CEREVISIAE MUTANTS DEFECTIVE IN THE START EVENT OF CELL DIVISION

Genetics ◽  
1980 ◽  
Vol 95 (3) ◽  
pp. 561-577 ◽  
Author(s):  
Steven I Reed
Keyword(s):  
Cell Division ◽  
Genetic Analysis ◽  
Linkage Map ◽  
Nuclear Genes ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mating Pheromone ◽  
Preliminary Characterization ◽  
Complementation Groups ◽  
Selection Of

ABSTRACT Thirty-three temperature-sensitive mutations defective in the start event of the cell division cycle of Saccharomyces cereuisiae were isolated and subjected to preliminary characterization. Complementation studies assigned these mutations to four complementation groups, one of which, cdc28, has been described previously. Genetic analysis revealed that these complementation groups define single nuclear genes, unlinked to one another. One of the three newly identified genes, cdc37, has been located in the yeast linkage map on chromosome IV, two meiotic map units distal to hom2.—Each mutation produces stage-specific arrest of cell division at start, the same point where mating pheromone interrupts division. After synchronization at start by incubation at the restrictive temperature, the mutants retain the capacity to enlarge and to conjugate.

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

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