Characterisation of fission yeast DNA replication origins.

In many eukaryotic organisms the chromosomal origins of DNA replication (ORIs) are not characterised by a clearly defined consensus sequence. In this thesis using the fission yeast, for the first time I have carried out a genome-wide analysis to identify such ORIs during the mitotic and meiotic cell cycles. The data can be summarised as follows: a total of 401 ORIs were identified which were used 29 percent of the time during mitotic S-phase and were spaced every 31 kilobases (kb) on average. The same ORIs were used during pre-meiotic S-phase although with lower efficiency in most chromosomal regions. A further 503 potential ORIs were used less efficiently at eight percent of the time during mitotic S-phase. This totals 904 ORIs which were distributed at an average inter-origin distance of 14 kilobases (kb) throughout 12.5 megabases (Mb) of the three chromosomes of fission yeast. These data support the idea of a continuum of ORI activity. The 401 efficient ORI loci contained A+T-rich regions located between genes, and these intergenic regions were typically larger than average. ORIs were not defined by a strict sequence consensus but the presence of AT-hook binding sequences. When the initiation factors Cdc18 and Cdt1 were over-expressed, regions of DNA containing particularly efficient ORIs with exceptionally large AT-hook binding domains became over-amplified, suggesting that interactions between these factors and efficient ORIs may be important for the mechanism ensuring that an ORI only fires once in each S-phase

OriDB, the DNA replication origin database updated and extended

OriDB (http://www.oridb.org/) is a database containing collated genome-wide mapping studies of confirmed and predicted replication origin sites. The original database collated and curated Saccharomyces cerevisiae origin mapping studies. Here, we report that the OriDB database and web site have been revamped to improve user accessibility to curated data sets, to greatly increase the number of curated origin mapping studies, and to include the collation of replication origin sites in the fission yeast Schizosaccharomyces pombe. The revised database structure underlies these improvements and will facilitate further expansion in the future. The updated OriDB for S. cerevisiae is available at http://cerevisiae.oridb.org/ and for S. pombe at http://pombe.oridb.org/

Global Profiling of DNA Replication Timing and Efficiency Reveals that Efficient Replication/Firing Occurs Late during S-Phase in S. pombe

10.1371/journal.pone.0000722PLoS ONE2

Epigenetically-Inherited Centromere and Neocentromere DNA Replicates Earliest in S-Phase

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position

Modeling Inhomogeneous DNA Replication Kinetics

In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited

Novel features of ARS selection in budding yeast Lachancea kluyveri

<p>Abstract</p> <p>Background</p> <p>The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.</p> <p>Results</p> <p>In this study we have isolated and characterized autonomously replicating sequences (ARSs) in <it>Lachancea kluyveri </it>- a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that <it>L. kluyveri </it>ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in <it>Saccharomyces cerevisiae</it>. Moreover, compared with <it>S. cerevisiae </it>and <it>K. lactis</it>, the replication licensing machinery in <it>L. kluyveri </it>seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all <it>S. cerevisiae </it>ARSs tested and most <it>Kluyveromyces lactis </it>ARSs. In contrast, only about half of the <it>L. kluyveri </it>ARSs function in <it>S. cerevisiae </it>and less than 10% function in <it>K. lactis</it>.</p> <p>Conclusions</p> <p>Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.</p

The Fission Yeast Homeodomain Protein Yox1p Binds to MBF and Confines MBF-Dependent Cell-Cycle Transcription to G1-S via Negative Feedback

The regulation of the G1- to S-phase transition is critical for cell-cycle progression. This transition is driven by a transient transcriptional wave regulated by transcription factor complexes termed MBF/SBF in yeast and E2F-DP in mammals. Here we apply genomic, genetic, and biochemical approaches to show that the Yox1p homeodomain protein of fission yeast plays a critical role in confining MBF-dependent transcription to the G1/S transition of the cell cycle. The yox1 gene is an MBF target, and Yox1p accumulates and preferentially binds to MBF-regulated promoters, via the MBF components Res2p and Nrm1p, when they are transcriptionally repressed during the cell cycle. Deletion of yox1 results in constitutively high transcription of MBF target genes and loss of their cell cycle–regulated expression, similar to deletion of nrm1. Genome-wide location analyses of Yox1p and the MBF component Cdc10p reveal dozens of genes whose promoters are bound by both factors, including their own genes and histone genes. In addition, Cdc10p shows promiscuous binding to other sites, most notably close to replication origins. This study establishes Yox1p as a new regulatory MBF component in fission yeast, which is transcriptionally induced by MBF and in turn inhibits MBF-dependent transcription. Yox1p may function together with Nrm1p to confine MBF-dependent transcription to the G1/S transition of the cell cycle via negative feedback. Compared to the orthologous budding yeast Yox1p, which indirectly functions in a negative feedback loop for cell-cycle transcription, similarities but also notable differences in the wiring of the regulatory circuits are evident

Mathematical Modelling of DNA Replication Reveals a Trade-off between Coherence of Origin Activation and Robustness against Rereplication

Eukaryotic genomes are duplicated from multiple replication origins exactly once per cell cycle. In Saccharomyces cerevisiae, a complex molecular network has been identified that governs the assembly of the replication machinery. Here we develop a mathematical model that links the dynamics of this network to its performance in terms of rate and coherence of origin activation events, number of activated origins, the resulting distribution of replicon sizes and robustness against DNA rereplication. To parameterize the model, we use measured protein expression data and systematically generate kinetic parameter sets by optimizing the coherence of origin firing. While randomly parameterized networks yield unrealistically slow kinetics of replication initiation, networks with optimized parameters account for the experimentally observed distribution of origin firing times. Efficient inhibition of DNA rereplication emerges as a constraint that limits the rate at which replication can be initiated. In addition to the separation between origin licensing and firing, a time delay between the activation of S phase cyclin-dependent kinase (S-Cdk) and the initiation of DNA replication is required for preventing rereplication. Our analysis suggests that distributive multisite phosphorylation of the S-Cdk targets Sld2 and Sld3 can generate both a robust time delay and contribute to switch-like, coherent activation of replication origins. The proposed catalytic function of the complex formed by Dpb11, Sld3 and Sld2 strongly enhances coherence and robustness of origin firing. The model rationalizes how experimentally observed inefficient replication from fewer origins is caused by premature activation of S-Cdk, while premature activity of the S-Cdk targets Sld2 and Sld3 results in DNA rereplication. Thus the model demonstrates how kinetic deregulation of the molecular network governing DNA replication may result in genomic instability

A Comprehensive Genome-Wide Map of Autonomously Replicating Sequences in a Naive Genome

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins. The machinery that initiates DNA synthesis is highly conserved, but the sites where the replication initiation proteins bind have diverged significantly. Functional comparative genomics is an obvious approach to study the evolution of replication origins. However, to date, the Saccharomyces cerevisiae replication origin map is the only genome map available. Using an iterative approach that combines computational prediction and functional validation, we have generated a high-resolution genome-wide map of DNA replication origins in Kluyveromyces lactis. Unlike other yeasts or metazoans, K. lactis autonomously replicating sequences (KlARSs) contain a 50 bp consensus motif suggestive of a dimeric structure. This motif is necessary and largely sufficient for initiation and was used to dependably identify 145 of the up to 156 non-repetitive intergenic ARSs projected for the K. lactis genome. Though similar in genome sizes, K. lactis has half as many ARSs as its distant relative S. cerevisiae. Comparative genomic analysis shows that ARSs in K. lactis and S. cerevisiae preferentially localize to non-syntenic intergenic regions, linking ARSs with loci of accelerated evolutionary change

Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms