Short DNA fragments without sequence similarity are initiation sites for replication in the chromosome of the yeast Yarrowia lipolytica.
(1/2033)
We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity. (+info)
Defects in Saccharomyces cerevisiae protein phosphatase type I activate the spindle/kinetochore checkpoint.
(2/2033)
A conditional allele of type 1 protein phosphatase (glc7-129) in Saccharomyces cerevisiae causes first cycle arrest in G2/M, characterized by cells with a short spindle and high H1 kinase activity. Point-of-execution experiments indicate Glc7p function is required in G2/M just before anaphase for the completion of mitosis. Loss of the spindle/kinetochore checkpoint in glc7-129 cells abolishes the G2/M cell cycle arrest with a concomitant increase in chromosome loss and reduced viability. These results support a role for Glc7p in regulating kinetochore attachment to the spindle, an event monitored by the spindle/kinetochore checkpoint. (+info)
Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p.
(3/2033)
We have investigated the role of protein phosphorylation in regulation of Saccharomyces cerevisiae kinetochores. By use of phosphatase inhibitors and a type 1 protein phosphatase mutant (glc7-10), we show that the microtubule binding activity, but not the centromeric DNA-binding activity, of the kinetochore complex is regulated by a balance between a protein kinase and the type 1 protein phosphatase (PP1) encoded by the GLC7 gene. glc7-10 mutant cells exhibit low kinetochore-microtubule binding activity in vitro and a high frequency of chromosome loss in vivo. Specifically, the Ndc10p component of the centromere DNA-binding CBF3 complex is altered by the glc7-10 mutation; Ndc10p is hyperphosphorylated in glc7-10 extracts. Furthermore, addition of recombinant Ndc10p reconstitutes the microtubule-binding activity of a glc7-10 extract to wild-type levels. Finally, the glc7-10-induced mitotic arrest is abolished in spindle checkpoint mutants, suggesting that defects in kinetochore-microtubule interactions caused by hyperphosphorylation of kinetochore proteins activate the spindle checkpoint. (+info)
An in vitro system recapitulates chromatin remodeling at the PHO5 promoter.
(4/2033)
The Saccharomyces cerevisiae gene PHO5 is an excellent system with which to study regulated changes in chromatin structure. The PHO5 promoter is packaged into four positioned nucleosomes under repressing conditions; upon induction, the structure of these nucleosomes is altered such that the promoter DNA becomes accessible to nucleases. We report here the development and characterization of an in vitro system in which partially purified PHO5 minichromosomes undergo promoter chromatin remodeling. Several hallmarks of the PHO5 chromatin transition in vivo were reproduced in this system. Chromatin remodeling of PHO5 minichromosomes required the transcription factors Pho4 and Pho2, was localized to the promoter region of PHO5, and was independent of the chromatin-remodeling complex Swi-Snf. In vitro chromatin remodeling also required the addition of fractionated nuclear extract and hydrolyzable ATP. This in vitro system should serve as a useful tool for identifying the components required for this reaction and for elucidating the mechanism by which the PHO5 promoter chromatin structure is changed. (+info)
Chromatin remodeling and activation of chromosomal DNA replication by an acidic transcriptional activation domain from BRCA1.
(5/2033)
An increasing number of transcription factors have been shown to activate DNA replication. However, the underlying mechanism remains to be elucidated. Here it is shown that when tethered to a cellular replication origin, the acidic transcriptional activation domain of the breast cancer protein BRCA1 alters the local chromatin structure and stimulates chromosomal DNA replication. Cancer-predisposing mutations in BRCA1 that abolish transcriptional activation also prevent chromatin remodeling and activation of replication. Chromatin remodeling occurs even in the absence of a functional replication origin. Thus, increasing chromatin accessibility may be an important mechanism used by transcription factors to facilitate multiple nuclear processes. (+info)
Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy.
(6/2033)
Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is approximately 9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent approximately 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis. (+info)
Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth.
(7/2033)
Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomyces cerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol alpha. DNA2 alleles were isolated that cause either temperature sensitivity, sensitivity to alkylation damage, or both. The alkylation-sensitive alleles clustered in the helicase domain, including changes in residues required for helicase activity in related proteins. Additional mutations known or expected to destroy the ATPase and helicase activities of Dna2 were constructed and found to support growth on some media but to cause alkylation sensitivity. Only damage-sensitive alleles were lethal in combination with a ctf4 deletion. Full activity of the Dna2 helicase function is therefore not needed for viability, but is required for repairing damage and for tolerating loss of Ctf4. Arrest of dna2 mutants was RAD9 dependent, but deleting this checkpoint resulted in either no effect or suppression of defects, including the synthetic lethality with ctf4. Dna2 therefore appears to act in repair or lagging strand synthesis together with Pol alpha and Ctf4, in a role that is optimal with, but does not require, full helicase activity. (+info)
Base excision repair of N-methylpurines in a yeast minichromosome. Effects of transcription, dna sequence, and nucleosome positioning.
(8/2033)
Base excision repair of dimethyl sulfate induced N-methylpurines (NMPs) was measured in a yeast minichromosome that has a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene, and varied regions of chromatin structure. Removal rates of NMPs varied dramatically (>20-fold) at different sites along three selected fragments encompassing a total length of 1775 base pairs. Repair of NMPs was not coupled to transcription, because the transcribed strands of HIS3 and induced GAL1:URA3 were not repaired faster than the nontranscribed strands. However, the repair rate of NMPs was significantly affected by the nearest neighbor nucleotides. Slow repair occurred at NMPs between purines, especially guanines, whereas fast repair occurred at NMPs between pyrimidines. NMPs between a purine and pyrimidine were repaired at moderate rates. Moreover, a rough correlation between nucleosome positions and repair rates exists in some but not all regions that were analyzed. (+info)