Tel1ATM dictates the replication timing of short yeast telomeres

Telomerase action is temporally linked to DNA replication. Although yeast telomeres are normally late replicating, telomere shortening leads to early firing of subtelomeric DNA replication origins. We show that double‐strand breaks flanked by short telomeric arrays cause origin firing early in S phase at late‐replicating loci and that this effect on origin firing time is dependent on the Tel1^ATM checkpoint kinase. The effect of Tel1^ATM on telomere replication timing extends to endogenous telomeres and is stronger than that elicited by Rif1 loss. These results establish that Tel1^ATM specifies not only the extent but also the timing of telomerase recruitment.     

Regulation of homologous recombination at telomeres in budding yeast

Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae.     

Time of replication of yeast centromeres and telomeres

The time of replication of centromeres and telomeres of the yeast S. cerevisiae was determined by performing Meselson-Stahl experiments with synchronized cells. The nine centromeres examined become hybrid in density early in S phase, eliminating the possibility that a delay in the replication of centromeres until mitosis is responsible for sister chromatid adherence and proper chromosome segregation at anaphase. The conserved sequence element Y', present at most telomeres, replicates late in S phase, as do the unique sequences adjacent to five specific telomeres. The early and late replication times of these structural elements may be either essential for their proper function or a consequence of some architectural feature of the chromosome.     

Mrc1 protects uncapped budding yeast telomeres from exonuclease EXO1

Mrc1 (Mediator of Replication Checkpoint 1) is a component of the DNA replication fork machinery and is necessary for checkpoint activation after replication stress. In this study, we addressed the role of Mrc1 at uncapped telomeres. Our experiments show that Mrc1 contributes to the vitality of both cdc13-1 and yku70Delta telomere capping mutants. Cells with telomere capping defects containing MRC1 or mrc1(AQ), a checkpoint defective allele, exhibit similar growth, suggesting growth defects of cdc13-1 mrc1Delta are not due to checkpoint defects. This is in accordance with Mrc1-independent Rad53 activation after telomere uncapping. Poor growth of cdc13-1 mutants in the absence of Mrc1 is a result of enhanced single stranded DNA accumulation at uncapped telomeres. Consistent with this, deletion of EXO1, encoding a nuclease that contributes to single stranded DNA accumulation after telomere uncapping, improves growth of cdc13-1 mrc1Delta strains and decreases ssDNA production. Our observations show that Mrc1, a core component of the replication fork, plays an important role in telomere capping, protecting from nucleases and checkpoint pathways.     

Organization of DNA sequences and replication origins at yeast telomeres

We have shown that the DNA sequences adjacent to the telomeres of Saccharomyces cerevisiae chromosomes are highly conserved and contain a high density of replication origins. The salient features of these telomeres can be summarized as follows. There are three moderately repetitive elements present at the telomeres: the 131 sequence (1 to 1.5 kb), the highly conserved Y sequence (5.2 kb), and the less conserved X sequence (0.3 to 3.75 kb). There is a high density of replication origins spaced about 6.7 kb apart at the telomeres. These replication origins are part of the X or the Y sequences. Some of the 131-Y repetitive units are tandemly arranged. The terminal sequence T (about 0.33 to 0.6 kb) is different from the 131, X, or Y sequences and is heterogeneous in length. The order of these sequences from the telomeric end towards the centromere is T-(Y-131)n-X-, where n ranges from 1 to no more than 4. Although these telomeric sequences are conserved among S. cerevisiae strains, they show striking divergence in certain closely related yeast species.     

DNA replication joins the revolution: whole-genome views of DNA replication in budding yeast

Replication origins, which are responsible for initiating the replication of eukaryotic chromosomal DNAs, are spaced at intervals of 40 to 200 kb. Although the sets of proteins that assemble at replication origins during G(1) to form pre-replicative complexes are highly conserved, the structures of replication origins varies from organism to organism. The identification of replication origins has been a labor-intensive task, requiring the analysis of chromosomal DNA replication intermediates. As a result, only a few replication origins have been identified and studied. In a pair of recently published papers, Raghuraman and colleagues and Wyrick, Aparicio and colleagues provide complementary microarray-based approaches to the identification of replication origins. These genome-wide views of DNA replication in Saccharomyces cerevisiae provide new insights into the way that the genome is duplicated and hold promise for the analysis of other genomes.     

Self-regulating model for control of replication origin firing in budding yeast

A major research area concentrates on understanding the regulation of replication origin firing. It is now appreciated that checkpoint signaling participates in this controlled process and that defects in such signaling systems affect genome integrity. Inhibition of replication origin firing is most obviously apparent under conditions of replication stress, but origin firing must also be regulated on a minute-by-minute basis as cells progress normally through an unabated S-phase. Here we summarize a straightforward model to account for how origin firing could be controlled by a self-regulating system.     

Stochastic association of neighboring replicons creates replication factories in budding yeast

Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.     

DNA replication stress is a determinant of chronological lifespan in budding yeast

The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans.     

Topoisomerase II inactivation prevents the completion of DNA replication in budding yeast

Type II topoisomerases are essential for resolving topologically entwined double-stranded DNA. Although anti-topoisomerase 2 (Top2) drugs are clinically important antibiotics and chemotherapies, to our knowledge, the mechanisms of cell killing by Top2 depletion and inactivation have never been directly compared. We show that depletion of Top2 protein from budding yeast cells prevents DNA decatenation during S phase. Cells complete DNA replication and enter the ensuing mitosis on schedule, suffering extensive chromosome missegregation. Cytokinesis through incompletely segregated chromosomes causes lethal DNA damage. By contrast, expression of catalytically inactive Top2 causes a stable G2 arrest requiring an intact DNA damage checkpoint. Checkpoint activation correlates with an inability to complete DNA replication, resulting in hypercatenated, gapped daughter DNA molecules. Thus, Top2 depletion and inactivation kill cells by different mechanisms, which has implications for understanding the nature of the catenation checkpoint, how DNA replication terminates, how anti-Top2 drugs work, and how new drugs might be designed.     

MRX protects telomeric DNA at uncapped telomeres of budding yeast cdc13-1 mutants

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.     

The positioning and dynamics of origins of replication in the budding yeast nucleus

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)-tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.     

Recombination can either help maintain very short telomeres or generate longer telomeres in yeast cells with weak telomerase activity

Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.     

Reciprocal association of the budding yeast ATM-related proteins Tel1 and Mec1 with telomeres in vivo

The phosphoinositide (PI)-3-kinase-related kinase (PIKK) family proteins Tel1p and Mec1p have been implicated in the telomere integrity of Saccharomyces cerevisiae. However, the mechanism of PIKK-mediated telomere length control remains unclear. Here, we show that Tel1p and Mec1p are recruited to the telomeres at specific times in the cell cycle in a mutually exclusive manner. In particular, Mec1p interacts with the telomeres during late S phase and is associated preferentially with shortened telomeres. We propose a model in which telomere integrity is maintained by the reciprocal association of PIKKs, and Mec1p acts as a sensor for structural abnormalities in the telomeres. Our study suggests a mechanistic similarity between telomere length regulation and DNA double-strand break repair, both of which are achieved by the direct association of PIKKs.     

Regulatory mechanism of the initiation step of DNA replication by CDK in budding yeast

Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. At the onset of chromosomal DNA replication, CDKs phosphorylate two replication proteins, Sld2 and Sld3, in budding yeast. Phosphorylated Sld2 and Sld3 enhance the formation of complexes with the BRCT (BRCA1 C-terminal)-containing replication protein Dpb11. The formation of these complexes is essential and sufficient for the CDK-dependent activation of the initiation of chromosomal DNA replication. Multiple phosphorylation of Sld2 by CDKs fine-tunes the process of complex formation. Here, we discussed the regulation of the initiation step of chromosomal DNA replication via CDK-dependent phosphorylation.     

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.     

Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast

Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S-phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S-phase is not known, but two kinase activities, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are continually required throughout the S-phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over-expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S-phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S-phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.