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 Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR‐primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C‐terminal tagged genes or for the exchange of the promoter and N‐terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C‐terminal epitope tags as well as nine different promoter substitutions in combination with N‐terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast‐optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N‐ and C‐terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost‐effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches. Copyright © 2004 John Wiley & Sons, Ltd.

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. Single-molecule 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.

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. G1-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 Clb5/6 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.

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 insir2Δ 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.

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.