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1.  GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome 
Time-resolved ChIP-chip can be utilized to monitor the genome-wide dynamics of the GINS complex, yielding quantitative information on replication fork movement.Replication forks progress at remarkably uniform rates across the genome, regardless of location.GINS progression appears to be arrested, albeit with very low frequency, at sites of highly transcribed genes.Comparison of simulation with data leads to novel biological insights regarding the dynamics of replication fork progression
In mitotic division, cells duplicate their DNA in S phase to ensure that the proper genetic material is passed on to their progeny. This process of DNA replication is initiated from several hundred specific sites, termed origins of replication, spaced across the genome. It is essential for replication to begin only after G1 and finish before the initiation of anaphase (Blow and Dutta, 2005; Machida et al, 2005). To ensure proper timing, the beginning stages of DNA replication are tightly coupled to the cell cycle through the activity of cyclin-dependent kinases (Nguyen et al, 2001; Masumoto et al, 2002; Sclafani and Holzen, 2007), which promote the accumulation of the pre-RC at the origins and initiate replication. Replication fork movement occurs subsequent to the firing of origins on recruitment of the replicative helicase and the other fork-associated proteins as the cell enters S phase (Diffley, 2004). The replication machinery itself (polymerases, PCNA, etc.) trails behind the helicase, copying the newly unwound DNA in the wake of the replication fork.
One component of the pre-RC, the GINS complex, consists of a highly conserved set of paralogous proteins (Psf1, Psf2, Psf3 and Sld5 (Kanemaki et al, 2003; Kubota et al, 2003; Takayama et al, 2003)). Previous work suggests that the GINS complex is an integral component of the replication fork and that its interaction with the genome correlates directly to the movement of the fork (reviewed in Labib and Gambus, 2007). Here, we used the GINS complex as a surrogate to measure features of the dynamics of replication—that is, to determine which origins in the genome are active, the timing of their firing and the rates of replication fork progression.
The timing of origin firing and the rates of fork progression have also been investigated by monitoring nascent DNA synthesis (Raghuraman et al, 2001; Yabuki et al, 2002). Origin firing was observed to occur as early as 14 min into the cell cycle and as late as 44 min (Raghuraman et al, 2001). A wide range of nucleotide incorporation rates (0.5–11 kb/min) were observed, with a mean of 2.9 kb/min (Raghuraman et al, 2001), whereas a second study reported a comparable mean rate of DNA duplication of 2.8±1.0 kb/min (Yabuki et al, 2002). In addition to these observations, replication has been inferred to progress asymmetrically from certain origins (Raghuraman et al, 2001). These data have been interpreted to mean that the dynamics of replication fork progression are strongly affected by local chromatin structure or architecture, and perhaps by interaction with the machineries controlling transcription, repair and epigenetic maintenance (Deshpande and Newlon, 1996; Rothstein et al, 2000; Raghuraman et al, 2001; Ivessa et al, 2003). In this study, we adopted a complementary ChIP-chip approach for assaying replication dynamics, in which we followed GINS complexes as they traverse the genome during the cell cycle (Figure 1). These data reveal that GINS binds to active replication origins and spreads bi-directionally and symmetrically as S phase progresses (Figure 3). The majority of origins appear to fire in the first ∼15 min of S phase. A small fraction (∼10%) of the origins to which GINS binds show no evidence of spreading (category 3 origins), although it remains possible that these peaks represent passively fired origins (Shirahige et al, 1998). Once an active origin fires, the GINS complex moves at an almost constant rate of 1.6±0.3 kb/min. Its movement through the inter-origin regions is consistent with that of a protein complex associated with a smoothly moving replication fork. This progression rate is considerably lower and more tightly distributed than those inferred from previous genome-wide measurements assayed through nascent DNA production (Raghuraman et al, 2001; Yabuki et al, 2002). Our study leads us to a different view of replication fork dynamics wherein fork progression is highly uniform in rate and little affected by genomic location.
In this work, we also observe a large number of low-intensity persistent features at sites of high transcriptional activity (e.g. tRNA genes). We were able to accurately simulate these features by assuming they are the result of low probability arrest of replication forks at these sites, rather than fork pausing (Deshpande and Newlon, 1996). The extremely low frequency of these events in wild-type cells suggests they are due to low probability stochastic occurrences during the replication process. It is hoped that future studies will resolve whether these persistent features indeed represent rare instances of fork arrest, or are the result of some alternative process. These may include, for example, the deposition of GINS complexes (or perhaps more specifically Psf2) once a pause has been resolved.
In this work, we have made extensive use of modeling to test a number of different hypotheses and assumptions. In particular, iterative modeling allowed us to infer that GINS progression is uniform and smooth throughout the genome. We have also demonstrated the potential of simulations for estimating firing efficiencies. In the future, extending such firing efficiency simulations to the whole genome should allow us to make correlations with chromosomal features such as nucleosome occupancy. Such correlations may help in determining factors that govern the probability of replication initiation throughout the genome.
Previous studies have led to a picture wherein the replication of DNA progresses at variable rates over different parts of the budding yeast genome. These prior experiments, focused on production of nascent DNA, have been interpreted to imply that the dynamics of replication fork progression are strongly affected by local chromatin structure/architecture, and by interaction with machineries controlling transcription, repair and epigenetic maintenance. Here, we adopted a complementary approach for assaying replication dynamics using whole genome time-resolved chromatin immunoprecipitation combined with microarray analysis of the GINS complex, an integral member of the replication fork. Surprisingly, our data show that this complex progresses at highly uniform rates regardless of genomic location, revealing that replication fork dynamics in yeast is simpler and more uniform than previously envisaged. In addition, we show how the synergistic use of experiment and modeling leads to novel biological insights. In particular, a parsimonious model allowed us to accurately simulate fork movement throughout the genome and also revealed a subtle phenomenon, which we interpret as arising from low-frequency fork arrest.
PMCID: PMC2858444  PMID: 20212525
cell cycle; ChIP-chip; DNA replication; replication fork; simulation
2.  Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome 
PLoS Computational Biology  2011;7(12):e1002322.
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.
Author Summary
Eukaryotic chromosomes replicate from multiple replication origins that fire at different times in S phase. The mechanisms that specify origin position and firing time and coordinate origins to ensure complete genome duplication are unclear. Previous studies proposed either that origins are arranged in temporally coordinated groups or fire independently of each other in a stochastic manner. Here, we have performed a quantitative analysis of human genome replication kinetics using a combination of DNA combing, which reveals local patterns of origin firing and replication fork progression on single DNA molecules, and massive sequencing of newly replicated DNA, which reveals the population-averaged replication timing profile of the entire genome. We show that origins are activated synchronously in large regions of uniform replication timing but more gradually in temporal transition regions and that the rate of origin firing increases as replication progresses. Large regions of unidirectional fork progression are abundant in embryonic stem cells but rare in differentiated cells. We propose a model in which replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA in a manner that explains the shape of the replication timing profile. These results provide a fundamental insight into the temporal regulation of mammalian genome replication.
PMCID: PMC3248390  PMID: 22219720
3.  Modeling genome-wide replication kinetics reveals a mechanism for regulation of replication timing 
We developed analytical models of DNA replication that include probabilistic initiation of origins, fork progression, passive replication, and asynchrony.We fit the model to budding yeast genome-wide microarray data probing the replication fraction and found that initiation times correlate with the precision of timing.We extracted intrinsic origin properties, such as potential origin efficiency and firing-time distribution, which cannot be done using phenomenological approaches.We propose that origin timing is controlled by stochastically activated initiators bound to origin sites rather than explicit time-measuring mechanisms.
The kinetics of DNA replication must be controlled for cells to develop properly. Although the biochemical mechanisms of origin initiations are increasingly well understood, the organization of initiation timing as a genome-wide program is still a mystery. With the advance of technology, researchers have been able to generate large amounts of data revealing aspects of replication kinetics. In particular, the use of microarrays to probe the replication fraction of budding yeast genome wide has been a successful first step towards unraveling the details of the replication program (Raghuraman et al, 2001; Alvino et al, 2007; McCune et al, 2008). On the surface, the microarray data shows apparent patterns of early and late replicating regions and seems to support the prevailing picture of eukaryotic replication—origins are positioned at defined sites and initiated at defined, preprogrammed times (Donaldson, 2005). Molecular combing, a single-molecule technique, however, showed that the initiation of origins is stochastic (Czajkowsky et al, 2008). Motivated by these conflicting viewpoints, we developed a model that is flexible enough to describe both deterministic and stochastic initiation.
We modeled origin initiation as probabilistic events. We first propose a model where each origin is allowed to have its distinct ‘firing-time distribution.' Origins that have well-determined initiation times have narrow distributions, whereas more stochastic origins have wider distributions. Similar models based on simulations have previously been proposed (Lygeros et al, 2008; Blow and Ge, 2009; de Moura et al, 2010); however, our model is novel in that it is analytic. It is much faster than simulations and allowed us, for the first time, to fit genome-wide microarray data and extract parameters that describe the replication program in unprecedented detail (Figure 2).
Our main result is this: origins that fire early, on average, have precisely defined initiation times, whereas origins that fire late, on average, do not have a well-defined initiation time and initiate throughout S phase. What kind of global controlling mechanism can account for this trend? We propose a second model where an origin is composed of multiple initiators, each of which fires independently and identically. A good candidate for the initiator is the minichromosome maintenance (MCM) complex, as it is found to be associated with origin firing and loaded in abundance (Hyrien et al, 2003). We show that the aforementioned relationship can be explained quantitatively if the earlier-firing origins have more MCM complexes. This model offers a new view of replication: controlled origin timing can emerge from stochastic firing and does not need an explicit time-measuring mechanism, a ‘clock.' This model provides a new, detailed, plausible, and testable mechanism for replication timing control.
Our models also capture the effects of passive replication, which is often neglected in phenomenological approaches (Eshaghi et al, 2007). There are two ways an origin site can be replicated. The site can be replicated by the origin binding to it but can also be passively replicated by neighboring origins. This complication makes it difficult to extract the intrinsic properties of origins. By modeling passive replication, we can separate the contribution from each origin and extract the potential efficiency of origins, i.e., the efficiency of the origin given that there is no passive replication. We found that while most origins are potentially highly efficient, their observed efficiency varies greatly. This implies that many origins, though capable of initiating, are often passively replicated and appear dormant. Such a design makes the replication process robust against replication stress such as fork stalling (Blow and Ge, 2009). If two approaching forks stall, normally dormant origins in the region, not being passively replicated, will initiate to replicate the gap.
With the advance of the microarray and molecular-combing technology, experiments have been done to probe many different types of cells, and large amounts of replication fraction data have been generated. Our model can be applied to spatiotemporally resolved replication fraction data for any organism, as the model is flexible enough to capture a wide range of replication kinetics. The analytical model is also much faster than simulation-based models. For these reasons, we believe that the model is a powerful tool for analyzing these large datasets. This work opens the possibility for understanding the replication program across species in more rigor and detail (Goldar et al, 2009).
Microarrays are powerful tools to probe genome-wide replication kinetics. The rich data sets that result contain more information than has been extracted by current methods of analysis. In this paper, we present an analytical model that incorporates probabilistic initiation of origins and passive replication. Using the model, we performed least-squares fits to a set of recently published time course microarray data on Saccharomyces cerevisiae. We extracted the distribution of firing times for each origin and found that the later an origin fires on average, the greater the variation in firing times. To explain this trend, we propose a model where earlier-firing origins have more initiator complexes loaded and a more accessible chromatin environment. The model demonstrates how initiation can be stochastic and yet occur at defined times during S phase, without an explicit timing program. Furthermore, we hypothesize that the initiators in this model correspond to loaded minichromosome maintenance complexes. This model is the first to suggest a detailed, testable, biochemically plausible mechanism for the regulation of replication timing in eukaryotes.
PMCID: PMC2950085  PMID: 20739926
DNA replication program; genome-wide analysis; microarray data; replication-origin efficiency; stochastic modeling
4.  Replication and Subnuclear Location Dynamics of the Immunoglobulin Heavy-Chain Locus in B-Lineage Cells 
Molecular and Cellular Biology  2002;22(13):4876-4889.
The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-Cα gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An ∼500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.
PMCID: PMC133899  PMID: 12052893
5.  Early activated replication origins within the cell cycle-regulated histone H4 genes in Physarum. 
Nucleic Acids Research  1999;27(10):2091-2098.
It was previously shown that the two members of the cell cycle-regulated histone H4 gene family, H4-1 and H4-2, are replicated at the onset of S phase in the naturally synchronous plasmodium of Physarum polycephalum, suggesting that they are flanked by replication origins. It was further shown that a DNA fragment upstream of the H4-1 gene is able to confer autonomous replication of a plasmid in the budding yeast. In this paper, we re-investigated replication of the unlinked Physarum histone H4 genes by mapping the replication origin of these two loci using alkaline agarose gel and neutral/neutral 2-dimensional agarose gel electrophoreses. We showed that the two replicons containing the H4 genes are simultaneously activated at the onset of S phase and we mapped an efficient, bidirectional replication origin in the vicinity of each gene. Our data demonstrated that the Physarum sequence that functions as an ARS in yeast is not the site of replication initiation at the H4-1 locus. We also observed a stalling of the rightward moving replication fork downstream of the H4-1 gene, in a region where transient topoisomerase II sites were previously mapped. Our results further extend the concept of replication/transcription coupling in Physarum to cell cycle-regulated genes.
PMCID: PMC148428  PMID: 10219081
6.  The One-Kilobase DNA Fragment Upstream of the ardC Actin Gene of Physarum polycephalum Is Both a Replicator and a Promoter 
Molecular and Cellular Biology  1999;19(5):3506-3514.
The 1-kb DNA fragment upstream of the ardC actin gene of Physarum polycephalum promotes the transcription of a reporter gene either in a transient-plasmid assay or as an integrated copy in an ectopic position, defining this region as the transcriptional promoter of the ardC gene (PardC). Since we mapped an origin of replication activated at the onset of S phase within this same fragment, we examined the pattern of replication of a cassette containing the PardC promoter and the hygromycin phosphotransferase gene, hph, integrated into two different chromosomal sites. In both cases, we show by two-dimensional agarose gel electrophoresis that an efficient, early activated origin coincides with the ectopic PardC fragment. One of the integration sites was a normally late-replicating region. The presence of the ectopic origin converted this late-replicating domain into an early-replicating domain in which replication forks propagate with kinetics indistinguishable from those of the native PardC replicon. This is the first demonstration that initiation sites for DNA replication in Physarum correspond to cis-acting replicator sequences. This work also confirms the close proximity of a replication origin and a promoter, with both functions being located within the 1-kb proximal region of the ardC actin gene. A more precise location of the replication origin with respect to the transcriptional promoter must await the development of a functional autonomously replicating sequence assay in Physarum.
PMCID: PMC84143  PMID: 10207074
7.  Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans 
PLoS Genetics  2014;10(4):e1004344.
Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-ACaCse4 levels at the programmed stall sites of early replicating centromeres.
Author Summary
The epigenetic mark of centromeres, CENP-A, is deposited in S phase in most yeasts by a mechanism that is not completely understood. Here, we identify two CEN7 flanking replication origins, ORI7-L1 and ORI7-RI, proximal to an early replicating centromere (CEN7) in a budding yeast Candida albicans. Replication forks starting from these origins stall randomly at CEN7 by the kinetochore that serves as a barrier to fork progression. We observe that centromeric fork stalling is reduced in absence of the HR proteins, Rad51 and Rad52, known to play a role in restarting stalled forks. Further, we demonstrate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. CENP-ACaCse4 levels are reduced in absence of Rad51 or Rad52, which results in disruption of the kinetochore structure. Here we propose a novel DNA replication-coupled mechanism mediated by HR proteins which epigenetically maintains centromere identity by regulating CENP-A deposition. A direct role of DNA repair proteins in centromere function offers insights into the mechanisms of centromere mis-regulation that leads to widespread aneuploidy in cancer cells.
PMCID: PMC3998917  PMID: 24762765
8.  Global Profiling of DNA Replication Timing and Efficiency Reveals that Efficient Replication/Firing Occurs Late during S-Phase in S. pombe 
PLoS ONE  2007;2(8):e722.
During S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins) that are enriched with AT-rich sequences, at various times. Current studies of genome-wide DNA replication profiles have focused on the DNA replication timing and origin location. However, the replication and/or firing efficiency of the individual origins on the genomic scale remain unclear.
Methodology/Principal Findings
Using the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. The increase in DNA copy number plotted as a function of time is approximated to the near-sigmoidal model, when considering the replication start and end timings at individual loci in cells released from HU-arrest. Replication efficiencies differ from origin to origin, depending on the origin's firing efficiency. We have found that DNA replication is inefficient early in S-phase, due to inefficient firing at origins. Efficient replication occurs later, attributed to efficient but late-firing origins. Furthermore, profiles of replication timing in cds1Δ cells are abnormal, due to the failure in resuming replication at the collapsed forks. The majority of the inefficient origins, but not the efficient ones, are found to fire in cds1Δ cells after HU removal, owing to the firing at the remaining unused (inefficient) origins during HU treatment.
Taken together, our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins. Additionally, checkpoint kinase Cds1p is required for maintaining the efficient replication/firing late in S-phase. We further propose that efficient late-firing origins are essential for ensuring completion of DNA duplication by the end of S-phase.
PMCID: PMC1934932  PMID: 17684567
9.  Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum. 
Molecular and Cellular Biology  1996;16(3):968-976.
We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmodium of the slime mold Physarum polycephalum. Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins. Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site-specific, bidirectional origin of replication is localized within the promoter region of the gene. We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins. Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions. Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P. polycephalum.
PMCID: PMC231079  PMID: 8622700
10.  Regulation of DNA Replication within the Immunoglobulin Heavy-Chain Locus During B Cell Commitment 
PLoS Biology  2012;10(7):e1001360.
The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus). The firing rate of origins shows a high degree of coordination across Igh domains that span tens to hundreds of kilobases, a phenomenon not observed in simple eukaryotes. Differences in domain sizes and firing rates determine the temporal order of replication. During B cell commitment, the expression of the B-cell-specific factor Pax5 sharply alters the temporal order of replication by modifying the rate of origin firing within various Igh domains (particularly those containing Pax5 binding sites). We propose that, within the Igh CH-3′RR domain, Pax5 is responsible for both establishing and maintaining high rates of origin firing, mostly by controlling events downstream of the assembly of pre-replication complexes.
Author Summary
Each time a mammalian cell duplicates its genome in preparation for cell division it activates thousands of so called “DNA origins of replication.” The timely and complete duplication of the genome depends on careful orchestration of origin activation, which is modified when cells differentiate to perform a specific function. We currently lack a universally accepted model of origin regulation that can explain the replication dynamics in complex eukaryotes. Here, we studied the mouse immunoglobulin heavy-chain locus, one of the antibody-encoding portions of the genome, where origins change activity when antibody-producing B cells differentiate in the bone marrow. We show that multiple aspects of DNA replication initiation, progression, and termination can be explained mathematically by the interplay between randomly firing origins and two independent variables: the speed of progression of replication forks and the firing rate of origins along the locus. The rate of origin firing varies extensively along the locus during B cell differentiation and, thus, is a dominant factor in establishing the temporal order of replication. A differentiation factor called Pax5 can alter the temporal order of replication by modifying the rate of origin firing across various parts of the locus.
PMCID: PMC3393677  PMID: 22807655
11.  USF Binding Sequences from the HS4 Insulator Element Impose Early Replication Timing on a Vertebrate Replicator 
PLoS Biology  2012;10(3):e1001277.
A combination of cis-regulatory elements can impose the formation of an early replicating domain in a naturally late replicating region and might constitute the basic unit of early replicating domains.
The nuclear genomes of vertebrates show a highly organized program of DNA replication where GC-rich isochores are replicated early in S-phase, while AT-rich isochores are late replicating. GC-rich regions are gene dense and are enriched for active transcription, suggesting a connection between gene regulation and replication timing. Insulator elements can organize independent domains of gene transcription and are suitable candidates for being key regulators of replication timing. We have tested the impact of inserting a strong replication origin flanked by the β-globin HS4 insulator on the replication timing of naturally late replicating regions in two different avian cell types, DT40 (lymphoid) and 6C2 (erythroid). We find that the HS4 insulator has the capacity to impose a shift to earlier replication. This shift requires the presence of HS4 on both sides of the replication origin and results in an advance of replication timing of the target locus from the second half of S-phase to the first half when a transcribed gene is positioned nearby. Moreover, we find that the USF transcription factor binding site is the key cis-element inside the HS4 insulator that controls replication timing. Taken together, our data identify a combination of cis-elements that might constitute the basic unit of multi-replicon megabase-sized early domains of DNA replication.
Author Summary
All eukaryotic organisms must duplicate their genome precisely once before cell division. This occurs according to an established temporal program during S-phase (when DNA synthesis takes place) of the cell cycle. In vertebrates, this program is regulated at the level of large chromosomal domains ranging from 200 kb to 2 Mb, but the molecular mechanisms that control the temporal firing order of animal replication origins are not clearly understood. Using the genetically tractable chicken DT40 cell system, we identified a minimal combination of cis-regulatory DNA elements that is able to shift the timing of a naturally “mid-late” replicated region to “mid-early.” This critical group of elements is composed of one strong replication origin flanked by binding sequences for the upstream stimulatory factor (USF) protein. The additional presence of a strongly transcribed gene shifted the region towards an even earlier replication time, suggesting cooperation between cis-elements when establishing temporal programs of replication. We speculate that USF binding sequences cooperate with sites of replication initiation and transcribed genes to promote the establishment of early replicating domains along vertebrate genomes.
PMCID: PMC3295818  PMID: 22412349
12.  The DNA Damage Response Pathway Contributes to the Stability of Chromosome III Derivatives Lacking Efficient Replicators 
PLoS Genetics  2010;6(12):e1001227.
In eukaryotic chromosomes, DNA replication initiates at multiple origins. Large inter-origin gaps arise when several adjacent origins fail to fire. Little is known about how cells cope with this situation. We created a derivative of Saccharomyces cerevisiae chromosome III lacking all efficient origins, the 5ORIΔ-ΔR fragment, as a model for chromosomes with large inter-origin gaps. We used this construct in a modified synthetic genetic array screen to identify genes whose products facilitate replication of long inter-origin gaps. Genes identified are enriched in components of the DNA damage and replication stress signaling pathways. Mrc1p is activated by replication stress and mediates transduction of the replication stress signal to downstream proteins; however, the response-defective mrc1AQ allele did not affect 5ORIΔ-ΔR fragment maintenance, indicating that this pathway does not contribute to its stability. Deletions of genes encoding the DNA-damage-specific mediator, Rad9p, and several components shared between the two signaling pathways preferentially destabilized the 5ORIΔ-ΔR fragment, implicating the DNA damage response pathway in its maintenance. We found unexpected differences between contributions of components of the DNA damage response pathway to maintenance of ORIΔ chromosome derivatives and their contributions to DNA repair. Of the effector kinases encoded by RAD53 and CHK1, Chk1p appears to be more important in wild-type cells for reducing chromosomal instability caused by origin depletion, while Rad53p becomes important in the absence of Chk1p. In contrast, RAD53 plays a more important role than CHK1 in cell survival and replication fork stability following treatment with DNA damaging agents and hydroxyurea. Maintenance of ORIΔ chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORIΔ chromosome stability. Thus, components of the DNA damage response pathway contribute to genome stability, not simply by detecting and responding to DNA template damage, but also by facilitating replication of large inter-origin gaps.
Author Summary
Loss of genome integrity underlies aspects of aging and human disease. During DNA replication, two parallel signaling pathways play important roles in the maintenance of genome integrity. One pathway detects DNA damage, while the other senses replication stress. Both pathways activate responses that include arrest of cell cycle progression, giving cells time to cope with the problem. These pathways have been defined by treating cells with compounds that induce either replication stress or DNA damage, but little is known about their roles during unperturbed DNA replication. They may be important when several adjacent replication origins fail to initiate and forks from flanking origins must replicate longer regions of DNA than normal to complete replication. We have used a derivative of budding yeast chromosome III lacking all efficient replication origins to identify mutants that preferentially destabilize this chromosome fragment, which mimics a chromosome with a large inter-origin gap. We found that the DNA damage response pathway, but not the replication stress response pathway, plays an important role in maintaining this fragment. The signal recognized in this case may be replisome failure rather than forks stalled at endogenous DNA damage.
PMCID: PMC2996327  PMID: 21151954
13.  Checkpoint independence of most DNA replication origins in fission yeast 
BMC Molecular Biology  2007;8:112.
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).
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.
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.
PMCID: PMC2235891  PMID: 18093330
14.  Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells 
Cell cycle (Georgetown, Tex.)  2009;8(19):3133-3148.
We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.
PMCID: PMC2829940  PMID: 19738421
DNA replication; S phase; origins; clusters; merging forks; fiber spreading; normal cells; cancer cells
15.  Quantitative, genome-wide analysis of eukaryotic replication initiation and termination 
Molecular cell  2013;50(1):123-135.
Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep-sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome; this permits the systematic analysis of initiation, origin efficiency, fork progression and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.
PMCID: PMC3628276  PMID: 23562327
16.  Do replication forks control late origin firing in Saccharomyces cerevisiae? 
Nucleic Acids Research  2011;40(5):2010-2019.
Recent studies of eukaryotic DNA replication timing profiles suggest that the time-dependent rate of origin firing, I(t), has a universal shape, which ensures a reproducible replication completion time. However, measurements of I(t) are based on population averages, which may bias the shape of the I(t) because of imperfect cell synchrony and cell-to-cell variability. Here, we measure the population-averaged I(t) profile from synchronized Saccharomyces cerevisiae cells using DNA combing and we extract the single-cell I(t) profile using numerical deconvolution. The single cell I(t) and the population-averaged I(t) extracted from DNA combing and replication timing profiles are similar, indicating a genome scale invariance of the replication process, and excluding cell-to-cell variability in replication time as an explanation for the shape of I(t). The single cell I(t) correlates with fork density in wild-type cells, which is specifically loosened in late S phase in the clb5Δ mutant. A previously proposed numerical model that reproduces the wild-type I(t) profile, could also describe the clb5Δ mutant I(t) once modified to incorporate the decline in CDK activity and the looser dependency of initiation on fork density in the absence of Clb5p. Overall, these results suggest that the replication forks emanating from early fired origins facilitate origin firing in later-replicating regions.
PMCID: PMC3300028  PMID: 22086957
17.  Origin activation and formation of single-strand TG1-3 tails occur sequentially in late S phase on a yeast linear plasmid. 
Molecular and Cellular Biology  1993;13(7):4057-4065.
In order to understand the mechanisms leading to the complete duplication of linear eukaryotic chromosomes, the temporal order of the events involved in replication of a 7.5-kb Saccharomyces cerevisiae linear plasmid called YLpFAT10 was determined. Two-dimensional agarose gel electrophoresis was used to map the position of the replication origin and the direction of replication fork movement through the plasmid. Replication began near the center of YLpFAT10 at the site in the 2 microns sequences that corresponds to the 2 microns origin of DNA replication. Replication forks proceeded bidirectionally from the origin to the ends of YLpFAT10. Thus, yeast telomeres do not themselves act as origins of DNA replication. The time of origin utilization on YLpFAT10 and on circular 2 microns DNA in the same cells was determined both by two-dimensional gel electrophoresis and by density transfer experiments. As expected, 2 microns DNA replicated in early S phase. However, replication of YLpFAT10 occurred in late S phase. Thus, the time of activation of the 2 microns origin depended upon its physical context. Density transfer experiments established that the acquisition of telomeric TG1-3 single-strand tails, a predicted intermediate in telomere replication, occurred immediately after the replication forks approached the ends of YLpFAT10. Thus, telomere replication may be the very last step in S phase.
PMCID: PMC359955  PMID: 8321213
18.  Replication Fork Reversal after Replication–Transcription Collision 
PLoS Genetics  2012;8(4):e1002622.
Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle.
Author Summary
Genomes are duplicated prior to cell division by DNA replication, and in all organisms replication impairment leads to chromosome instability. In bacteria, replication and transcription take place simultaneously, and in eukaryotes house-keeping genes are expressed during the S-phase; consequently, transcription is susceptible to impair replication progression. Here, we increase head-on replication–transcription collisions on the bacterial chromosome by inversion of a ribosomal operon (rrn). We show that only one recombination protein is required for growth when the rrn genes are highly expressed: the RecBCD complex, an exonuclease/recombinase that promotes degradation and RecA-dependent homologous recombination of linear DNA. In the absence of RecBCD, we observe linear DNA that ends in the collision region. This linear DNA is composed of only the origin-proximal region of the inverted rrn operon, indicating that it results from fork breakage. It is partly RuvABC-dependent (i.e. produced by the E. coli Holliday junction resolvase), indicating that blocked forks are reversed. The linear DNA ends up at the inverted rrn locus only if the RecJ exonuclease is inactivated; otherwise it is degraded, with major products ending in other upstream rrn operons, indicating that DNA degradation is slowed down by ribosomal operon sequences.
PMCID: PMC3320595  PMID: 22496668
19.  Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories 
The Journal of Cell Biology  2010;191(7):1285-1297.
At low levels of replication stress, Chk1 favors resolving problems at stalled replication forks over initiating origin firing in unreplicated areas of the genome.
Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3–related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.
PMCID: PMC3010067  PMID: 21173116
20.  Modeling Inhomogeneous DNA Replication Kinetics 
PLoS ONE  2012;7(3):e32053.
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.
PMCID: PMC3296702  PMID: 22412853
21.  DNA Replication Forks Pause at Silent Origins near the HML Locus in Budding Yeast 
Molecular and Cellular Biology  2001;21(15):4938-4948.
Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex (ORC) and a heterochromatin structure, while the functions of ARS303 and ARS320 are not known. In this work, we discovered replication fork pause sites at the HML ARS cluster and ARS301 by analyzing DNA replication intermediates from the chromosome via two-dimensional gel electrophoresis. The replication fork pause at the HML ARS cluster was independent of cis- and trans-acting mutations that abrogate transcriptional silencing at HML. Deletion of the HML ARS cluster led to loss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HML ARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the pause but activated the silent origin at the HML ARS cluster in a minority of cells. Delaying the time of fork arrival at HML led to the elimination of the pause sites at the HML ARS cluster and at the copy of ARS305 inserted in place of the cluster. Loss of the pause sites was accompanied by activation of the silent origins in the majority of cells. Thus, replication fork movement near HML pauses at a silent origin which is competent for replication initiation but kept silent through Orc2p, a component of the replication initiator. Possible functions for replication fork pause sites in checkpoints, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed.
PMCID: PMC87221  PMID: 11438651
22.  Asynchronous Replication, Mono-Allelic Expression, and Long Range Cis-Effects of ASAR6 
PLoS Genetics  2013;9(4):e1003423.
Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.
Author Summary
Mammalian chromosomes are duplicated every cell cycle during a precise temporal DNA replication program. Thus, every chromosome contains regions that are replicated early and other regions that are replicated late during each S phase. Most of the genes, present in two copies on homologous chromosomes, replicate synchronously during each S phase. Exceptions to this rule are genes located on X chromosomes, genetically imprinted genes, and genes subject to allelic exclusion. Thus, all mono-allelically expressed genes are subject to asynchronous replication, where one allele replicates before the other. Perhaps the best-studied example of asynchronous replication in mammals occurs during X inactivation in female cells. A large non-coding RNA gene called XIST, located within the X inactivation center, controls the transcriptional silencing and late replication of the inactive X chromosome. We have identified a locus on human chromosome 6 that shares many characteristics with XIST. This chromosome 6 locus encodes a large intergenic non-coding RNA gene, ASAR6, which displays random mono-allelic expression, asynchronous replication, and controls the mono-allelic expression of other genes on chromosome 6. Our work supports a model in which all mammalian chromosomes contain similar cis-acting loci that function to ensure proper chromosome replication, mitotic condensation, mono-allelic expression, and stability of individual chromosomes.
PMCID: PMC3617217  PMID: 23593023
23.  Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region. 
Molecular and Cellular Biology  1997;17(10):6167-6174.
DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.
PMCID: PMC232467  PMID: 9315677
24.  Organization of DNA replication in Physarum polycephalum. Attachment of origins of replicons and replication forks to the nuclear matrix. 
Nucleic Acids Research  1983;11(4):1181-1195.
We have investigated the attachment of the DNA to the nuclear matrix during the division cycle of the plasmodial slime mold Physarum polycephalum. The DNA of plasmodia was pulse labelled at different times during the S phase and the label distribution was studied by graded DNase digestion of the matrix-DNA complexes prepared from nuclei isolated by extraction with 2 M NaCl. Pulse labelled DNA was preferentially recovered from the matrix bound residual DNA at any time of the S phase. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the G2 phase and the subsequent S phase. The occurrence of the pulse label in the matrix associated DNA regions was transiently elevated at the onset of the subsequent S phase. Label incorporated at the end of the S phase was located at DNA regions which, in the G2 phase, were preferentially released from the matrix by DNase treatment. From the results and previously reported data on the distribution of attachment sites it can be concluded that origins of replicons or DNA sites very close to them are attached to the matrix during the entire nuclear cycle. The data further indicate that initiations of DNA replication occur at the same origins in successive S phases. Replicating DNA is bound to the matrix, in addition, by the replication fork or a region close to it. This binding is loosened after completion of the replication.
PMCID: PMC325785  PMID: 6828380
25.  Damage-Induced Phosphorylation of Sld3 is Important to Block Late Origin Firing 
Nature  2010;467(7314):479-483.
Origins of replication are activated throughout S-phase such that some origins fire early and others fire late to ensure that each chromosome is completely replicated in a timely fashion. However, in response to DNA damage or replication fork stalling, eukaryotic cells block activation of unfired origins. Human cells derived from patients with ataxia telangiectasia are deficient in this process due to the lack of a functional ataxia-telegiectasia mutated (ATM) kinase and elicit Radio-resistant DNA synthesis (RDS)1–3 following γ-irradiation2. This effect is conserved in budding yeast, as yeast cells lacking the related kinase Mec1 (ATR) also fail to inhibit DNA synthesis in the presence of DNA damage4. This intra-S-phase checkpoint actively regulates DNA synthesis by inhibiting the firing of late replicating origins, and this inhibition requires both Mec1 and the downstream checkpoint kinase Rad53 (Chk2)5,6. However, the Rad53 substrate(s) whose phosphorylation is required to mediate this function remained unknown. Here, we show that the replication initiation protein Sld3 is phosphorylated by Rad53, and that this phosphorylation, along with phosphorylation of the Cdc7 kinase regulatory subunit Dbf4, blocks late origin firing. Upon exposure to DNA damaging agents, cells expressing nonphosphorylatable alleles of SLD3 and DBF4 (SLD3-m25 and dbf4-m25, respectively) proceed through S-phase faster than wild-type cells by inappropriately firing late origins of replication. SLD3-m25 dbf4-m25 cells grow poorly in the presence of the replication inhibitor hydroxyurea (HU) and accumulate multiple Rad52 foci. Moreover, SLD3-m25 dbf4-m25 cells are delayed in recovering from transient blocks to replication and subsequently arrest at the DNA damage checkpoint. These data suggest that the intra-S-phase checkpoint functions to block late origin firing in adverse conditions to prevent genomic instability and maximize cell survival.
PMCID: PMC3393088  PMID: 20865002

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