Mapping of Replication Origins in the X Inactivation Center of Vole Microtus levis Reveals Extended Replication Initiation Zone

DNA replication initiates at specific positions termed replication origins. Genome-wide studies of human replication origins have shown that origins are organized into replication initiation zones. However, only few replication initiation zones have been described so far. Moreover, few origins were mapped in other mammalian species besides human and mouse. Here we analyzed pattern of short nascent strands in the X inactivation center (XIC) of vole Microtus levis in fibroblasts, trophoblast stem cells, and extraembryonic endoderm stem cells and confirmed origins locations by ChIP approach. We found that replication could be initiated in a significant part of XIC. We also analyzed state of XIC chromatin in these cell types. We compared origin localization in the mouse and vole XIC. Interestingly, origins associated with gene promoters are conserved in these species. The data obtained allow us to suggest that the X inactivation center of M. levis is one extended replication initiation zone.     

Timely binding of IHF and Fis to DARS2 regulates ATP–DnaA production and replication initiation

In Escherichia coli, the ATP-bound form of DnaA (ATP–DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP–DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP–DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP–DnaA was fully active in replication initiation and underwent DnaA–ATP hydrolysis. ADP–DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP–DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP–DnaA production, thereby promoting timely initiation. Moreover, we show that IHF–DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP–DnaA and replication initiation in coordination with the cell cycle and growth phase.     

The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro.

Fis protein participates in the normal control of chromosomal replication in Escherichia coli. However, the mechanism by which it executes its effect is largely unknown. We demonstrate an inhibitory influence of purified Fis protein on replication from oriC in vitro. Fis inhibits DNA synthesis equally well in replication systems either dependent upon or independent of RNA polymerase, even when the latter is stimulated by the presence of HU or IHF. The extent of inhibition by Fis is modulated by the concentrations of DnaA protein and RNA polymerase; the more limiting the amounts of these, the more severe the inhibition by Fis. Thus, the level of inhibition seems to depend on the ease with which the open complex can be formed. Fis-mediated inhibition of DNA replication does not depend on a functional primary Fis binding site between DnaA boxes R2 and R3 in oriC, as mutations that cause reduced binding of Fis to this site do not affect the degree of inhibition. The data presented suggest that Fis prevents formation of an initiation-proficient structure at oriC by forming an alternative, initiation-preventive complex. This indicates a negative role for Fis in the regulation of replication initiation.     

Deletion of the datA site does not affect once-per-cell-cycle timing but induces rifampin-resistant replication

In Escherichia coli, three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the datA site. The datA site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in ΔdatA cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the datA site is not required to prevent reinitiation and limit initiation of replication to once per generation. The datA site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.     

Negative feedback for DARS2–Fis complex by ATP–DnaA supports the cell cycle-coordinated regulation for chromosome replication

In Escherichia coli, the replication initiator DnaA oscillates between an ATP- and an ADP-bound state in a cell cycle-dependent manner, supporting regulation for chromosome replication. ATP–DnaA cooperatively assembles on the replication origin using clusters of low-affinity DnaA-binding sites. After initiation, DnaA-bound ATP is hydrolyzed, producing initiation-inactive ADP–DnaA. For the next round of initiation, ADP–DnaA binds to the chromosomal locus DARS2, which promotes the release of ADP, yielding the apo-DnaA to regain the initiation activity through ATP binding. This DnaA reactivation by DARS2 depends on site-specific binding of IHF (integration host factor) and Fis proteins and IHF binding to DARS2 occurs specifically during pre-initiation. Here, we reveal that Fis binds to an essential region in DARS2 specifically during pre-initiation. Further analyses demonstrate that ATP–DnaA, but not ADP–DnaA, oligomerizes on a cluster of low-affinity DnaA-binding sites overlapping the Fis-binding region, which competitively inhibits Fis binding and hence the DARS2 activity. DiaA (DnaA initiator-associating protein) stimulating ATP–DnaA assembly enhances the dissociation of Fis. These observations lead to a negative feedback model where the activity of DARS2 is repressed around the time of initiation by the elevated ATP–DnaA level and is stimulated following initiation when the ATP–DnaA level is reduced.     

A criterion for using chloramphenicol to define different processes in the initiation of DNA synthesis in bacteria

A criterion is proposed which must be met in order to demonstrate that two different proteins are required for the initiation of DNA synthesis, with each protein being synthesized at a different time prior to initiation, and with the synthesis of each protein having a different sensitivity to inhibition by chloramphenicol. Specifically, as the CM concentration is continuously varied, there must be a discontinuous variation in the number of origins able to be initiated at each concentration. In the light of this criterion, I suggest that there is no firm evidence for the proposal that there exist two different proteins which are required for initiation of DNA replication in Escherichia coli.     

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

Time to Be Versatile: Regulation of the Replication Timing Program in Budding Yeast

Eukaryotic replication origins are activated at different times during the S phase of the cell cycle, following a temporal program that is stably transmitted to daughter cells. Although the mechanisms that control initiation at the level of individual origins are now well understood, much less is known on how cells coordinate replication at hundreds of origins distributed on the chromosomes. In this review, we discuss recent advances shedding new light on how this complex process is regulated in the budding yeast Saccharomyces cerevisiae. The picture that emerges from these studies is that replication timing is regulated in cis by mechanisms modulating the chromatin structure and the subnuclear organization of origins. These mechanisms do not affect the licensing of replication origins but determine their ability to compete for limiting initiation factors, which are recycled from early to late origins throughout the length of the S phase.     

Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, Cdc45-Mcm-GINS

Eukaryotic DNA replication is initiated at multiple origins of replication, where many replication proteins assemble under the control of the cell cycle [1]. A key process of replication initiation is to convert inactive Mcm2-7 to active Cdc45-Mcm-GINS (CMG) replicative helicase [2]. However, it is not known whether the CMG assembly would automatically activate its helicase activity and thus assemble the replisome. Mcm10 is an evolutionally conserved essential protein required for the initiation of replication [3, 4]. Although the roles of many proteins involved in the initiation are understood, the role of Mcm10 remains controversial [5-9]. To characterize Mcm10 in more detail, we constructed budding yeast cells bearing a degron-fused Mcm10 protein that can be efficiently degraded in response to auxin. In the absence of Mcm10, a stable CMG complex was assembled at origins. However, subsequent translocation of CMG, replication protein A loading to origins, and the intra-S checkpoint activation were severely diminished, suggesting that origin unwinding is defective. We also found that Mcm10 associates with origins during initiation in an S-cyclin-dependent kinase- and Cdc45-dependent manner. Thus, Mcm10 plays an essential role in functioning of the CMG replicative helicase independent of assembly of a stable CMG complex at origins.     

Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically

In rapidly growing Escherichia coli cells replication cycles overlap and initiation occurs at multiple replication origins (oriCs). All origins within a cell are initiated essentially in synchrony and only once per cell cycle. Immediate re-initiation of new origins is avoided by sequestration, a mechanism dependent on the SeqA protein and Dam methylation of GATC sites in oriC. Here, GATC sites in oriC were changed to GTTC. This reduced the sequestration to essentially the level found in SeqA-less cells. The mutant origins underwent re-initiation, showing that the GATC sites in oriC are required for sequestration. Each re-initiation eventually gave rise to a cell containing an extra nucleoid. The three-nucleoid cells displayed one asymmetrically placed FtsZ-ring and divided into a two-nucleoid cell and a one-nucleoid cell. The three nucleoid-cells thus divided into three daughters by two consecutive divisions. The results show that extra rounds of replication cause extra daughter cells to be formed prematurely. The fairly normal mutant growth rate and size distribution show, however, that premature rounds of replication, chromosome segregation, and cell division are flexibly accommodated by the existing cell cycle controls.     

Drunken-cell footprints: nuclease treatment of ethanol-permeabilized bacteria reveals an initiation-like nucleoprotein complex in stationary phase replication origins.

The nucleoprotein complex formed on oriC, the Escherichia coli replication origin, is dynamic. During the cell cycle, high levels of the initiator DnaA and a bending protein, IHF, bind to oriC at the time of initiation of DNA replication, while binding of Fis, another bending protein, is reduced. In order to probe the structure of nucleoprotein complexes at oriC in more detail, we have developed an in situ footprinting method, termed drunken-cell footprinting, that allows enzymatic DNA modifying reagents access to intracellular nucleoprotein complexes in E.coli, after a brief exposure to ethanol. With this method, we observed in situ binding of Fis to oriC in exponentially growing cells, and binding of IHF to oriC in stationary cells, using DNase I and Bst NI endonuclease, respectively. Increased binding of DnaA to oriC in stationary phase was also noted. Because binding of DnaA and IHF results in unwinding of oriC in vitro, P1 endonuclease was used to probe for intracellular unwinding of oriC. P1 cleavage sites, localized within the 13mer unwinding region of oriC ', were dramatically enhanced in stationary phase on wild-type origins, but not on mutant versions of oriC unable to unwind. These observations suggest that most oriC copies become unwound during stationary phase, forming an initiation-like nucleoprotein complex.     

Involvement of Fis protein in replication of the Escherichia coli chromosome.

We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.     

Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12.

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.     

Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. Quiescent cells contained very low levels of replication initiation factors, and their entry into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase.     

Genetic and physical mapping of DNA replication origins in Haloferax volcanii

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.     

Genetic interaction of RAD53 protein kinase with histones is important for DNA replication

Studies in budding yeast suggest the protein kinase Rad53 plays novel roles in controlling initiation of DNA replication and in maintaining cellular histone levels, and these roles are independent of Rad53-mediated regulation of the checkpoint and of nucleotide levels. In order to elucidate the role of Rad53 in replication initiation, we isolated a novel allele of RAD53, rad53-rep, that separates the checkpoint function of RAD53 from the DNA replication function. rad53-rep mutants display a chromosome loss phenotype that is suppressed by increased origin dosage, providing further evidence that Rad53 plays a role in the initiation of DNA replication. Deletion of the major histone H3–H4 pair suppresses rad53-rep-cdc7-1 synthetic lethality, suggesting Rad53''s functions in degradation of excess cellular histone and in replication initiation are related. Rad53-rep is active as a protein kinase yet fails to interact with origins of replication and like the rad53Δ mutant, the rad53-rep mutant accumulates excess soluble histones, and it is sensitive to histone dosage. In contrast, a checkpoint defective allele of RAD53 with mutations in both FHA domains, binds origins and growth of this mutant is unaffected by histone dosage. Based on these observations, we hypothesize that the origin binding and the histone degradation activities of Rad53 are central to its function in DNA replication and are independent of its checkpoint functions. We propose a model in which Rad53 acts as a “nucleosome buffer”, interacting with origins of replication to prevent the binding of excess histones to origin DNA and to maintain proper chromatin configuration.Key words: DNA replication, Rad53, histones, checkpoint, origins of replication