Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast

No abstract

The functions of the Cdc28 protein kinase in DNA replication and mitosis in Saccharomyces cerevisiae are thought to be determined by the type of cyclin subunit with which it is associated. Gl-specific cyclins encoded by CLN1, CLN2, and CLN3 are required for entry into the cell cycle (Start) and thereby for S phase, whereas G2-specific B-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 are required for mitosis. We describe a new family of B-type cyclin genes, CLB5 and CLB6, whose transcripts appear in late G1 along with those of CLN1, CLN2, and many genes required for DNA replication. Deletion of CLB6 has little or no effect, but deletion of CLB5 greatly extends S phase, and deleting both genes prevents the timely initiation of DNA replication. Transcription of CLB5 and CLB6 is normally dependent on Cln activity, but ectopic CLB5 expression allows cells to proliferate in the absence of Cln cyclins. Thus, the kinase activity associated with C165/6 and not with Cln cyclins may be responsible for S-phase entry. Clb5 also has a function, along with Clb3 and Clb4, in the formation of mitotic spindles. Our observation that CLB5 is involved in the initiation of both S phase and mitosis suggests that a single primordial B-type cyclin might have been sufficient for regulating the cell cycle of the common ancestor of many, if not all, eukaryotes.

The six main minichromosome maintenance proteins (Mcm2-7), which presumably constitute the core of the replicative DNA helicase, are present in chromatin in large excess relative to the number of active replication forks. To evaluate the relevance of this apparent surplus of Mcm2-7 complexes in human cells, their levels were down-regulated by using RNA interference. Interestingly, cells continued to proliferate for several days after the acute (>90%) reduction of Mcm2-7 concentration. However, they became hypersensitive to DNA replication stress, accumulated DNA lesions, and eventually activated a checkpoint response that prevented mitotic division. When this checkpoint was abrogated by the addition of caffeine, cells quickly lost viability, and their karyotypes revealed striking chromosomal aberrations. Singlemolecule analyses revealed that cells with a reduced concentration of Mcm2-7 complexes display normal fork progression but have lost the potential to activate ''dormant'' origins that serve a backup function during DNA replication. Our data show that the chromatin-bound ''excess'' Mcm2-7 complexes play an important role in maintaining genomic integrity under conditions of replicative stress.DNA combing ͉ DNA replication ͉ origin licensing R apidly proliferating cells start to prepare for DNA replication several hours before the actual S-phase, with the assembly of prereplication complexes (pre-RCs) at origins in telophase and early G 1 . Pre-RC assembly, also referred to as ''origin licensing,'' consists in the recruitment of Mcm2-7 protein complexes by initiator proteins ORC, CDC6, and CDT1. ORC and CDC6 likely constitute a structural module with ATPase activity that opens and closes the ring-shaped MCM hexamer, facilitating its topological engagement with the DNA, whereas CDT1 cooperates in the loading reaction as a molecular chaperone (reviewed in refs. 1 and 2). Different lines of evidence indicate that Mcm2-7 constitute the core of the replicative DNA helicase in eukaryotic cells in association with CDC45 and the GINS complex (3, 4).The maximum number of origins available in the subsequent S-phase is predetermined at the licensing stage, because additional pre-RCs cannot be assembled later in the cell cycle because of the inhibitory activity of the S, G 2 and M-phase cyclin-dependent kinases. This regulation establishes a temporal alternation of origin licensing and firing that is important to prevent DNA overreplication. In yeast, blending the licensing and firing periods by deletion of the CDK inhibitor Sic1 or by overexpression of the G 1 cyclin Cln2, greatly increased genomic instability (5, 6). In human cells, premature expression of Cyclin E during mitosis and G 1 interfered with the association of MCM proteins with chromatin and at the same time promoted the firing of the limited number of licensed origins, effectively accelerating the G 1 -S transition (7). Nevertheless, cells continued to proliferate under these challenging conditions and accumulated karyotypic defects (8). These results are ...

G(1) cell cycle regulators are often mutated in cancer, but how this causes genomic instability is unclear. Here we show that yeast lacking the CDK inhibitor Sic1 initiate DNA replication from fewer origins, have an extended S phase, and inefficiently separate sister chromatids during anaphase. This leads to double-strand breaks (DSBs) in a fraction of sic1 cells as evidenced by the accumulation of Ddc1 foci and a 575-fold increase in gross chromosomal rearrangements. Both S and M phase defects are rescued by delaying S-CDK activation, indicating that Sic1 promotes origin licensing in late G(1) by preventing the untimely activation of CDKs. We propose that precocious CDK activation causes genomic instability by altering the dynamics of S phase, which then hinders normal chromosome segregation.

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication.

How eukaryotes specify their replication origins is an important unanswered question. Here, we analyze the replicative organization of yeast rDNA, which consists of ∼ 150 identical repeats, each containing a potential origin. Using DNA combing and single-molecule imaging, we show that functional rDNA origins are clustered and interspersed with large domains where initiation is silenced. This repression is largely mediated by the Sir2p histone-deacetylase. Increased origin firing in sir2⌬ mutants leads to the accumulation of circular rDNA species, a major determinant of yeast aging. We conclude that rDNA replication is regulated epigenetically and that Sir2p may promote genome stability and longevity by suppressing replication-dependent rDNA recombination. Received April 5, 2002; revised version accepted July 19, 2002. Eukaryotic chromosomes are replicated from multiple origins, which are activated sequentially throughout the S phase of the cell cycle. In the yeast Saccharomyces cerevisiae, as probably in other eukaryotes, most origins are not used every cell cycle, some of them being totally silent in normal growth conditions (Fangman and Brewer 1991;Pasero and Schwob 2000). A growing body of evidence indicates that chromosomal context is a major determinant of origin activity (Cimbora and Groudine 2001;Heun et al. 2001;Méchali 2001;Gerbi and Bielinsky 2002). However, how chromatin structure modulates origin activity is still poorly understood, and the biological significance of this regulation remains to be established.The ribosomal DNA cluster of S. cerevisiae, which consists of 100-200 identical copies of a 9.1-kb unit, is an interesting model system to address these questions. Indeed, every unit contains a potential origin of DNA replication (Fig. 1A,B), but only ∼ 20% of these elements were shown to be active in a single cell cycle (Linskens and Huberman 1988;Fangman and Brewer 1991). Because sequences are identical, epigenetic mechanisms likely control origin activity in the rDNA. However, owing to its repeated nature, the organization of replicons in this locus has been difficult to address using conventional approaches and leads to divergent interpretations (Walmsley et al. 1984;Saffer and Miller 1986).Here we use novel technologies based on BrdU incorporation in yeast and DNA combing to visualize replication origins on single rDNA molecules. Their distribution is nonrandom and dependent on the conserved histone deacetylase Sir2p, which is known to silence transcription and extend life span (Defossez et al. 2001;Gasser and Cockell 2001). Because yeast aging is linked to rDNA replication via the formation of extrachromosomal rDNA circles (Guarente 2000), our results suggest that Sir2p may promote genome stability and yeast longevity by modulating the number and distribution of rDNA replication origins. Results and DiscussionWe present here the first quantitative analysis of the distribution of replication origins in the repeated rDNA array of S. cerevisiae. A combination of bromodeoxyuridine (BrdU) in...

Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G 1 -phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G 1 phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC).