The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

In budding yeast, TEL1 encodes a protein closely related to ATM. Xrs2 is an Nbs1 homolog and forms a complex with Mre11 and Rad50. We show here that Tel1 associ ates with double-strand breaks (DSBs) through a mechanism dependent on the C terminus of Xrs2. Although Xrs2 is required for the DNA degradation at DSBs, the C-terminal Xrs2 truncation does not affect the degradation. Tel1 and the C terminus of Xrs2 are similarly involved in cell survival and Rad53 phosphorylation after DNA damage. Our findings suggest that the Tel1 association with DNA lesions is required for the activation of DNA damage responses. To ensure accurate transmission of genetic information, cells have evolved surveillance mechanisms that monitor genomic integrity and activate a variety of DNA damage responses, including cell-cycle arrest and alterations in gene expression (Zhou and Elledge 2000). DNA damage responses are initiated through the ATM family proteins, which are large protein kinases with similarity in the catalytic domain to phosphatidylinositol 3-kinases (Zhou and Elledge 2000). This family includes ATM (mammals), ATR (mammals), Mec1 (Saccharomyces cerevisiae), Tel1 (S. cerevisiae), and Rad3 (Schizosaccharomyces pombe). Each of these proteins falls into two subgroups based on homology; ATM is related most closely to Tel1, whereas ATR is more closely related to Mec1 and Rad3.In the budding yeast S. cerevisiae, Mec1 plays a critical role in the DNA damage response pathway, whereas Tel1 plays a minor role (Morrow et al. 1995;Sanchez et al. 1996). Mec1 physically interacts with Lcd1 (also called Ddc2 and Pie1), a protein that exhibits limited homology to the mammalian ATRIP and S. pombe Rad26 protein (Edwards et al. 1999;Paciotti et al. 2000;Rouse and Jackson 2000;Cortez et al. 2001;Wakayama et al. 2001). Both Mec1 and Lcd1 are recruited to sites of DNA damage, suggesting that the Mec1-Lcd1 complex may act as a primary sensor of DNA damage Melo et al. 2001;Rouse and Jackson 2002). RAD53 encodes a protein kinase related to the mammalian Chk2 protein (Zhou and Elledge 2000). Rad53 functions downstream of Mec1 and Tel1, and is activated and phosphorylated after DNA damage (Sanchez et al. 1996;Sun et al. 1996;Usui et al. 2001;Nakada et al. 2003). Recent observations suggest that Rad9 interacts physically with Rad53 to modulate its kinase activity Schwartz et al. 2002). Activation of the Rad53 pathway results in cell-cycle arrest and transcriptional induction of genes required for damage repair.The Mre11 complex consists of two highly conserved proteins, Mre11 and Rad50, and a third protein, either Xrs2 in budding yeast or Nbs1 in mammals (D'Amours and Jackson 2002). The Mre11 complex has been most extensively characterized in budding yeast, and it is implicated in two different repair processes of doublestrand breaks (DSBs): homologous recombination and nonhomologous end joining. In addition to DSB processing, the Mre11 complex is required for mediating the cellular response to DNA damage. Studies of human cells have demonstrated that ...

No abstract

We have cloned four cyclin-B homologs from Saccharomyces cerevisiae, CLB1-CLB4, using the polymerase chain reaction and low stringency hybridization approaches. These genes form two classes based on sequence relatedness: CLB1 and CLB2 show highest homology to the Schizosaccharomyces pombe cyclin-B homolog cdcl3 involved in the initiation of mitosis, whereas CLB3 and CLB4 are more highly related to the S. pombe cyclin-B homolog cigl, which appears to play a role in G1 or S phase. CLB1 and CLB2 mRNA levels peak late in the cell cycle, whereas CLB3 and CLB4 are expressed earlier in the cell cycle but peak later than the Gl-specific cyclin, CLN1. Analysis of null mutations suggested that the CLB genes exhibit some degree of redundancy, but clbl,2 and clb2,3 cells were inviable. Using clbl,2,3,4 cells rescued by conditional overproduction of CLB1, we showed that the CLB genes perform an essential role at the G2/M-phase transition, and also a role in S phase. CLB genes also appear to share a role in the assembly and maintenance of the mitotic spindle. Taken together, these analyses suggest that CLB1 and CLB2 are crucial for mitotic induction, whereas CLB3 and CLB4 might participate additionally in DNA replication and spindle assembly.

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.

Pheromone‐stimulated yeast cells and haploid gpa1 deletion mutants arrest their cell cycle in G1. Overexpression of a novel gene called MSG5 suppresses this inhibition of cell division. Loss of MSG5 function leads to a diminished adaptive response to pheromone. Genetic analysis indicates that MSG5 acts at a stage where the protein kinases STE7 and FUS3 function to transmit the pheromone‐induced signal. Since loss of MSG5 function causes an increase in FUS3 enzyme activity but not STE7 activity, we propose that MSG5 impinges on the pathway at FUS3. Sequence analysis suggests that MSG5 encodes a protein tyrosine phosphatase. This is supported by the finding that recombinant MSG5 has phosphatase activity in vitro and is able to inactivate autophosphorylated FUS3. Thus MSG5 might stimulate recovery from pheromone by regulating the phosphorylation state of FUS3.

Summary Chromosome ends, known as telomeres, have to be distinguished from DNA double-strand breaks (DSBs) that activate the DNA damage checkpoint. In budding yeast, the ATM homolog Tel1 associates preferentially with short telomeres and promotes telomere addition. Here we show that the telomeric proteins Rif1 and Rif2 attenuate Tel1 recruitment to DNA ends through distinct mechanisms. Both Rif1 and Rif2 inhibit the localization of Tel1, but not the Mre11-Rad50-Xrs2 (MRX) complex, to adjacent DNA ends. Rif1 function is weaker at short telomeric repeats compared with Rif2 function, and is partly dependent on Rif2. Rif2 competes with Tel1 for binding to the C-terminus of Xrs2. Once Tel1 is delocalized, MRX does not associate efficiently with Rap1-covered DNA ends. These results reveal a mechanism by which telomeric DNA sequences mask DNA ends from Tel1 recognition for the regulation of telomere length.

In response to DNA damage, eukaryotic cells activate checkpoint pathways that arrest cell cycle progression and induce the expression of genes required for DNA repair. In budding yeast, the homothallic switching (HO) endonuclease creates a site-specific double-strand break at the mating type (MAT) locus. Continuous HO expression results in the phosphorylation of Rad53, which is dependent on products of the ataxia telangiectasia mutated-related MEC1 gene and other checkpoint genes, including DDC1, RAD9, and RAD24. Chromatin immunoprecipitation experiments revealed that the Ddc1 protein associates with a region near the MAT locus after HO expression. Ddc1 association required Rad24 but not Mec1 or Rad9. Mec1 also associated with a region near the cleavage site after HO expression, but this association is independent of Ddc1, Rad9, and Rad24. Thus, Mec1 and Ddc1 are recruited independently to sites of DNA damage, suggesting the existence of two separate mechanisms involved in recognition of DNA damage.