A variable fork rate affects timing of origin firing and S phase dynamics in Saccharomyces cerevisiae

Activation (in the following referred to as firing) of replication origins is a continuous and irreversible process regulated by availability of DNA replication molecules and cyclin-dependent kinase activities, which are often altered in human cancers. The temporal, progressive origin firing throughout S phase appears as a characteristic replication profile, and computational models have been developed to describe this process. Although evidence from yeast to human indicates that a range of replication fork rates is observed experimentally in order to complete a timely S phase, those models incorporate velocities that are uniform across the genome. Taking advantage of the availability of replication profiles, chromosomal position and replication timing, here we investigated how fork rate may affect origin firing in budding yeast. Our analysis suggested that patterns of origin firing can be observed from a modulation of the fork rate that strongly correlates with origin density. Replication profiles of chromosomes with a low origin density were fitted with a variable fork rate, whereas for the ones with a high origin density a constant fork rate was appropriate. This indeed supports the previously reported correlation between inter-origin distance and fork rate changes. Intriguingly, the calculated correlation between fork rate and timing of origin firing allowed the estimation of firing efficiencies for the replication origins. This approach correctly retrieved origin efficiencies previously determined for chromosome VI and provided testable prediction for other chromosomal origins. Our results gain deeper insights into the temporal coordination of genome duplication, indicating that control of the replication fork rate is required for the timely origin firing during S phase.     

Visualization of altered replication dynamics after DNA damage in human cells

Eukaryotic cells respond to DNA damage within the S phase by activating an intra-S checkpoint: a response that includes reducing the rate of DNA synthesis. In yeast cells this can occur via checkpoint-dependent inhibition of origin firing and stabilization of ongoing forks, together with a checkpoint-independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. We have developed strategies based on DNA fiber labeling that allow the quantitative assessment of rates of replication fork movement, origin firing, and fork stalling throughout the genome by examining large numbers of individually labeled replication forks. We show that exposing S phase cells to ionizing radiation induces a transient block to origin firing but does not affect fork rate or fork stalling. Alkylation damage by methyl methane sulfonate causes a slowing of fork movement and a high rate of fork stalling, in addition to inducing a block to new origin firing. Nucleotide depletion by hydroxyurea also reduces replication fork rate and increases stalling; moreover, in contrast to a recent report, we show that hydroxyurea induces a strong block to new origin firing. The DNA fiber labeling strategy provides a powerful new approach to analyze the dynamics of DNA replication in a perturbed S phase.     

Brc1 links replication stress response and centromere function

Protection of genome integrity depends on the coordinated activities of DNA replication, DNA repair, chromatin assembly and chromosome segregation mechanisms. DNA lesions are detected by the master checkpoint kinases ATM (Tel1) and ATR (Rad3/Mec1), which phosphorylate multiple substrates, including a C-terminal SQ motif in histone H2A or H2AX. The 6-BRCT domain protein Brc1, which is required for efficient recovery from replication fork arrest and collapse in fission yeast, binds phospho-histone H2A (γH2A)-coated chromatin at stalled and damaged replication forks. We recently found that Brc1 co-localizes with γH2A that appears in pericentromeric heterochromatin during S-phase. Our studies indicate that Brc1 contributes to the maintenance of pericentromeric heterochromatin, which is required for efficient chromosome segregation during mitosis. Here, we review these studies and present additional results that establish the functional requirements for the N-terminal BRCT domains of Brc1 in the replication stress response and resistance to the microtubule destabilizing drug thiabendazole (TBZ). We also identify the nuclear localization signal (NLS) in Brc1, which closely abuts the C-terminal pair of BRCT domains that form the γH2A-binding pocket. This compact arrangement of localization domains may be a shared feature of other γH2A-binding proteins, including Rtt107, PTIP and Mdc1.     

The CDK regulators Cdh1 and Sic1 promote efficient usage of DNA replication origins to prevent chromosomal instability at a chromosome arm

Robustness and completion of DNA replication rely on redundant DNA replication origins. Reduced efficiency of origin licensing is proposed to contribute to chromosome instability in CDK-deregulated cell cycles, a frequent alteration in oncogenesis. However, the mechanism by which this instability occurs is largely unknown. Current models suggest that limited origin numbers would reduce fork density favouring chromosome rearrangements, but experimental support in CDK-deregulated cells is lacking. We have investigated the pattern of origin firing efficiency in budding yeast cells lacking the CDK regulators Cdh1 and Sic1. We show that each regulator is required for efficient origin activity, and that both cooperate non-redundantly. Notably, origins are differentially sensitive to CDK deregulation. Origin sensitivity is independent on normal origin efficiency, firing timing or chromosomal location. Interestingly, at a chromosome arm, there is a shortage of origin firing involving active and dormant origins, and the extent of shortage correlates with the severity of CDK deregulation and chromosome instability. We therefore propose that CDK deregulation in G1 phase compromises origin redundancy by decreasing the number of active and dormant origins, leading to origin shortage and increased chromosome instability.     

Histone acetylation regulates the time of replication origin firing

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.     

SIRT1 Deacetylates TopBP1 and Modulates Intra-S-Phase Checkpoint and DNA Replication Origin Firing

SIRT1, the mammalian homolog of yeast Sir2, is a founding member of a family of 7 protein and histone deacetylases that are involved in numerous biological functions. Previous studies revealed that SIRT1 deficiency results in genome instability, which eventually leads to cancer formation, yet the underlying mechanism is unclear. To investigate this, we conducted a proteomics study and found that SIRT1 interacted with many proteins involved in replication fork protection and origin firing. We demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. Mechanistically, SIRT1 deacetylates and affects the activity of TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Our study demonstrated that ectopic over-expression of the deacetylated form of TopBP1 in SIRT1 mutant cells repressed replication origin firing, while the acetylated form of TopBP1 lost this function. Thus, SIRT1 acts upstream of TopBP1 and plays an essential role in maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint.     

Transitions in replication timing in a 340 kb region of human chromosomal R-Band 1p36.1

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.     

High-resolution analysis of DNA replication domain organization across an R/G-band boundary.

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.     

Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin

The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.     

Replication timing properties across the pseudoautosomal region boundary and cytogenetic band boundaries on human distal Xp

The establishment of human chromosomal regions as distinct and characteristic domains has been demonstrated by the reproducible banding patterns observed on metaphase chromosomes as a result of various staining techniques. Although the exact molecular properties responsible for the patterns are not well understood, a general correlation has been established between the time of replication of a particular region of DNA and its banding characteristics. Using a replication timing assay based on fluorescence in situ hybridization patterns, we investigated replication timing properties across chromosomal regions with potentially distinct chromatin properties. Relative replication timing values were determined using cosmid DNA probes around the pseudoautosomal region boundary in Xp22.3 and the cytogenetic band boundary regions surrounding Xp22.2. Although we observed replication timing domains that were generally consistent with cytogenetic banding patterns, we did not find sharp replication timing boundaries at either the pseudoautosomal region boundary or at the cytogenetic band boundaries. Received: 6 September 1997; in revised form: 16 December 1997 / Accepted: 5 January 1998     

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.     

Checkpoint independence of most DNA replication origins in fission yeast

Background

In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2).

Results

Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.

Conclusion

The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.     

Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics.     

A model for the spatiotemporal organization of DNA replication in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

DNA replication in eukaryotes is considered to proceed according to a precise program in which each chromosomal region is duplicated in a defined temporal order. However, recent studies reveal an intrinsic temporal disorder in the replication of yeast chromosome VI. Here we provide a model of the chromosomal duplication to study the temporal sequence of origin activation in budding yeast. The model comprises four parameters that influence the DNA replication system: the lengths of the chromosomes, the explicit chromosomal positions for all replication origins as well as their distinct initiation times and the replication fork migration rate. The designed model is able to reproduce the available experimental data in form of replication profiles. The dynamics of DNA replication was monitored during simulations of wild type and randomly perturbed replication conditions. Severe loss of origin function showed only little influence on the replication dynamics, so systematic deletions of origins (or loss of efficiency) were simulated to provide predictions to be tested experimentally. The simulations provide new insights into the complex system of DNA replication, showing that the system is robust to perturbation, and giving hints about the influence of a possible disordered firing. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis

We propose a model for heterochromatin assembly that links DNA methylation with histone methylation and DNA replication. The hypomethylated Arabidopsis mutants ddm1 and met1 were used to investigate the relationship between DNA methylation and chromatin organization. Both mutants show a reduction of heterochromatin due to dispersion of pericentromeric low-copy sequences away from heterochromatic chromocenters. DDM1 and MET1 control heterochromatin assembly at chromocenters by their influence on DNA maintenance (CpG) methylation and subsequent methylation of histone H3 lysine 9. In addition, DDM1 is required for deacetylation of histone H4 lysine 16. Analysis of F(1) hybrids between wild-type and hypomethylated mutants revealed that DNA methylation is epigenetically inherited and represents the genomic imprint that is required to maintain pericentromeric heterochromatin.