Universal Temporal Profile of Replication Origin Activation in Eukaryotes

Although replication proteins are conserved among eukaryotes, the sequence requirements for replication initiation differ between species. In all species, however, replication origins fire asynchronously throughout S phase. The temporal program of origin firing is reproducible in cell populations but largely probabilistic at the single-cell level. The mechanisms and the significance of this program are unclear. Replication timing has been correlated with gene activity in metazoans but not in yeast. One potential role for a temporal regulation of origin firing is to minimize fluctuations in replication end time and avoid persistence of unreplicated DNA in mitosis. Here, we have extracted the population-averaged temporal profiles of replication initiation rates for S. cerevisiae, S. pombe, D. melanogaster, X. laevis and H. sapiens from genome-wide replication timing and DNA combing data. All the profiles have a strikingly similar shape, increasing during the first half of S phase then decreasing before its end. A previously proposed minimal model of stochastic initiation modulated by accumulation of a recyclable, limiting replication-fork factor and fork-promoted initiation of new origins, quantitatively described the observed profiles without requiring new implementations

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

International audienceDNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated

A Simple Model for the Influence of Meiotic Conversion Tracts on GC Content

A strong correlation between GC content and recombination rate is observed in many eukaryotes, which is thought to be due to conversion events linked to the repair of meiotic double-strand breaks. In several organisms, the length of conversion tracts has been shown to decrease exponentially with increasing distance from the sites of meiotic double-strand breaks. I show here that this behavior leads to a simple analytical model for the evolution and the equilibrium state of the GC content of sequences devoid of meiotic double-strand break sites. In the yeast Saccharomyces cerevisiae, meiotic double-strand breaks are practically excluded from protein-coding sequences. A good fit was observed between the predictions of the model and the variations of the average GC content of the third codon position (GC3) of S. cerevisiae genes. Moreover, recombination parameters that can be extracted by fitting the data to the model coincide with experimentally determined values. These results thus indicate that meiotic recombination plays an important part in determining the fluctuations of GC content in yeast coding sequences. The model also accounted for the different patterns of GC variations observed in the genes of Candida species that exhibit a variety of sexual lifestyles, and hence a wide range of meiotic recombination rates. Finally, the variations of the average GC3 content of human and chicken coding sequences could also be fitted by the model. These results suggest the existence of a widespread pattern of GC variation in eukaryotic genes due to meiotic recombination, which would imply the generality of two features of meiotic recombination: its association with GC-biased gene conversion and the quasi-exclusion of meiotic double-strand breaks from coding sequences. Moreover, the model points out to specific constraints on protein fragments encoded by exon terminal sequences, which are the most affected by the GC bias

Schematics illustrating the computation of asymmetry indices.

<p>(A) Definition of the four strand segments. (B) Four simple cases are shown for different positions <i>x</i>. Segments synthesized as leading and lagging strands are shown as blue and red lines, respectively. The small, black rectangles represent coding sequences with their transcriptional orientation given by the associated arrows. The large, black arrowheads indicate the orientation of the replication forks.</p

Variations of GC3 content in <i>S. cerevisiae</i> protein-coding sequences.

<p>The mean GC3 contents (A) or (B) of 66-codon segments are plotted as a function of the positions or relative to the ATG or to the stop codon, respectively, on which the segments are centered. For a given position or , and are averaged over classes of genes binned by their lengths. The genes of set 1 (, blue dotted line) contain 3 to 5 66-codon segments, the genes of set 2 (, blue dashed line) contain 6 to 8 segments, the genes of set 3 (, blue dot-dash line) contain 9 to 11 segments, the genes of set 4 (, blue long-dash line) contain 12 to 14 segments, and the genes of set 5 (, blue solid line) contain at least 15 segments. Only the average values for the segments common to all the genes of a given set are plotted (<i>i.e.</i> only the segments corresponding to the shortest genes of the set). The thick, red, solid lines represent the mean or averaged over all genes containing at least one 66-codon segment (). The thin, red, solid lines represent the mean theoretical values or averaged over all genes containing at least one 66-codon segment and whose values of and could be determined (). and were calculated as functions of , , and using Equations 7 and 8 (and the estimates of , , and given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016109#pone-0016109-t001" target="_blank">Table 1</a>), and averaged over segments with the same position or , respectively.</p

Asymmetry Indices for Analysis and Prediction of Replication Origins in Eukaryotic Genomes

<div><p>DNA replication was recently shown to induce the formation of compositional skews in the genomes of the yeasts <em>Saccharomyces cerevisiae</em> and <em>Kluyveromyces lactis</em>. In this work, I have characterized further GC and TA skew variations in the vicinity of <em>S. cerevisiae</em> replication origins and termination sites, and defined asymmetry indices for origin analysis and prediction. The presence of skew jumps at some termination sites in the <em>S. cerevisiae</em> genome was established. The majority of <em>S. cerevisiae</em> replication origins are marked by an oriented consensus sequence called ACS, but no evidence could be found for asymmetric origin firing that would be linked to ACS orientation. Asymmetry indices related to GC and TA skews were defined, and a global asymmetry index <em>I_GC,TA</em> was described. <em>I_GC,TA</em> was found to strongly correlate with origin efficiency in <em>S. cerevisiae</em> and to allow the determination of sets of intergenes significantly enriched in origin loci. The generalized use of asymmetry indices for origin prediction in naive genomes implies the determination of the direction of the skews, <em>i.e.</em> the identification of which strand, leading or lagging, is enriched in G and which one is enriched in T. Recent work indicates that in <em>Candida albicans</em> and in several related species, centromeres contain early and efficient replication origins. It has been proposed that the skew jumps observed at these positions would reflect the activity of these origins, thus allowing to determine the direction of the skews in these genomes. However, I show here that the skew jumps at <em>C. albicans</em> centromeres are not related to replication and that replication-associated GC and TA skews in <em>C. albicans</em> have in fact the opposite directions of what was proposed.</p> </div

The Spindle Assembly Checkpoint Regulates the Phosphorylation State of a Subset of DNA Checkpoint Proteins in Saccharomyces cerevisiae

The DNA and the spindle assembly checkpoints play key roles in maintaining genomic integrity by coordinating cell responses to DNA lesions and spindle dysfunctions, respectively. These two surveillance pathways seem to operate mostly independently of one another, and little is known about their potential physiological connections. Here, we show that in Saccharomyces cerevisiae, the activation of the spindle assembly checkpoint triggers phosphorylation changes in two components of the DNA checkpoint, Rad53 and Rad9. These modifications are independent of the other DNA checkpoint proteins and are abolished in spindle checkpoint-defective mutants, hinting at specific functions for Rad53 and Rad9 in the spindle damage response. Moreover, we found that after UV irradiation, Rad9 phosphorylation is altered and Rad53 inactivation is accelerated when the spindle checkpoint is activated, which suggests the implication of the spindle checkpoint in the regulation of the DNA damage response

Origin efficiency correlates with the global asymmetry index <i>I_GC,TA</i> in <i>Saccharomyces cerevisiae</i>.

<p>(A,B) Box plots displaying the differences in <i>I_GC,TA</i> values between replication origins annotated as confirmed, likely, and dubious (A) or as chromosomically active and inactive (B). The horizontal lines show the median values. The bottoms and tops of the boxes show the 25th and the 75th percentiles, respectively. (C,D) <i>I_GC,TA</i> values are plotted as a function of origin efficiency and average replication time, respectively. The dashed lines correspond to least square fits.</p

Variations of asymmetry indices around replication origins (A) and termination sites (B) in <i>Saccharomyces cerevisiae</i>.

<p>The average values of <i>I_GC,cod</i> (thick, red line), <i>I_TA,cod</i> (thick, blue line), <i>I_GC,int</i> (thin, red line) and <i>I_TA,int</i> (thin, blue line) were computed every 500 bp using a window length <i>L</i> = 10 kb. Position 0 corresponds to the position of the extended ACS sequence 5′-(T/A)(T/G)TTTAT(G/A)TTT(T/A)(G/C)(T/G)T-3′ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045050#pone.0045050-Nieduszynski2" target="_blank">[32]</a> for origins (A) and to the midpoint of termination regions (B). All origins were oriented according to their ACS, as indicated by the arrow in (A).</p

The initial slopes of and are correlated with the values of and for <i>S. cerevisiae</i> genes.

<p>The mean GC3 contents (A) or (B) of 66-codon segments are plotted as a function of the positions or relative to the ATG or to the stop codon, respectively, on which the segments are centered. and are averaged over classes of genes sorted by their lengths and their values of and . The green curves correspond to the genes containing 9 to 11 66-codon segments, the blue curves, to the genes containing 12 to 14 segments and the red curves, to the genes containing at least 15 segments. For each set of genes, the solid curves represent the genes with high values of either or , and the dashed curves represent the genes with low values of either or .</p