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 ...