Replication of polyoma DNA in isolated nuclei: analysis of replication fork movement.

The movement of replication forks during polyoma DNA synthesis in isolated nuclei was analyzed by digesting newly synthesized DNA with the restriction endonuclease HpaII which cleaves polyoma DNA into eight unique fragments. The terminus of in vitro DNA synthesis was identified by cleaving newly completed molecules with HpaII. The distribution of label in the restriction fragments showed that the in vitro DNA synthesis was bidirectional and had the normal terminus of replication. Analysis of replicative intermediates pulse-labeled in vitro further suggested that DNA synthesis in isolated nuclei is an ordered process similar to replication in intact cells. Replication forks moved with a constant rate from the origin towards the terminus of replication. The nonlinear course of the DNA synthesis reaction in the isolated nuclei seems to result from the random inactivation of replication forks rather than a decrease in the rate of fork movement. During the in vitro synthesis a replication fork could maximally synthesize a DNA chain about 1,000 nucleotides long. The results suggest that some replication forks might be initiated in vitro at the origin of replication.     

In vitro polyoma DNA synthesis: asymmetry of short DNA chains.

The kinetics of annealing of the separated strands of the polyoma DNA Hpa II restriction fragments 1 and 2 to an excess of purified short DNA chains isolated from in vitro pulse-labeled replicating polyoma DNA were determined. The results indicate that for each growing fork, the DNA strand which must grow discontinuously is represented about 4 times as frequently in the population of short DNA chains as the strand which could replicate continuously. In addition, the absolute concentration of short DNA chains in the two growing forks is approximately the same. The average size of the short DNA chains from the continuous strand was shown to be very similar to that of the short DNA chains from the discontinuous strand. We conclude that polyoma DNA replication in vitro proceeds by a predominantly semi-discontinuous mechanism.     

Polyoma virus DNA replication is semi-discontinuous.

In marked contrast to simian virus 40 (SV40), polyoma virus (PyV) has been reported to replicate discontinuously on both arms of replication forks. In an effort to clarify the relationship between the mechanisms of DNA replication in these closely related viruses, the distribution of RNA-primed DNA chains at replication forks was examined concurrently in PyV and SV40 replicating DNA purified from virus-infected cells. About one third of PyV DNA chains contained 7 to 9 ribonucleotides covalently linked to their 5'-end. A similar fraction of DNA chains from replicating SV40 DNA contained an oligoribonucleotide that was 6 to 9 residues long and began with either (p)ppA or (p)ppG. Greater than 80% of PyV or SV40 RNA-primed DNA chains hybridized specifically to the retrograde template. Moreover, at least 95% of the RNA-primed DNA chains from either PyV or SV40 whose initiation sites could be mapped to unique nucleotide locations originated from the retrograde template. Therefore, PyV and SV40 DNA replication forks are essentially the same; DNA synthesis is discontinuous predominantly, if not exclusively, on the retrograde template.     

Hydroxyurea-Induced Accumulation of Short Fragments During Polyoma DNA Replication I. Characterization of Fragments

Hydroxyurea treatment of 3T6 mouse fibroblast cells infected with polyoma virus resulted within 15 min in more than a 20-fold reduction of the rate of both viral and cellular DNA synthesis. After the initial rapid inhibition, the rate of DNA synthesis remained essentially constant for at least 2 h. In the inhibited cells viral DNA accumulated as short chains with a sedimentation coefficient of about 4S (hydroxyurea fragments). A variable proportion of these fragments was released from the template strands when the viral DNA was extracted by the Hirt procedure. Reannealing experiments demonstrated that hydroxyurea fragments were polyoma-specific and probably synthesized on both parental strands at the replication forks.     

Intermediate in SV40 DNA Chain Growth

PREVIOUS studies of the DNA replication of simian virus 40 (SV40), an oncogenic member of the papoyavirus group, have been concerned with separation and characterization of replicative intermediates^1–4. Circular replicating intermediates have been identified for SV40^1–3, as well as for the similar replication system of polyoma viral DNA5,6. The replicative intermediates of SV40 DNA have been observed by electron microscopy to contain two forks, three branches and no free ends^1–3 as is the case for the circular replicating molecules of polyoma, bacteriophage λ⁷, Escherichia coli⁸ and colicin E1 in mini-cells^9,10. An important property of replicative intermediates of SV40 DNA that has also been observed in replicating molecules of colicin E1¹⁰ is that most molecules contain a superhelical region in the unreplicated portion of the molecule¹.     

Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

In Escherichia coli, the SeqA protein binds specifically to GATC sequences which are methylated on the A of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork. We show that this is, indeed, the case by using global ChIP on Chip analysis of SeqA in cells synchronized regarding DNA replication. To assess hemimethylation, we developed the first genome-wide method for methylation analysis in bacteria. Since loss of the SeqA protein affects growth rate only during rapid growth when cells contain multiple replication forks, a comparison of rapid and slow growth was performed. In cells with six replication forks per chromosome, the two old forks were found to bind surprisingly little SeqA protein. Cell cycle analysis showed that loss of SeqA from the old forks did not occur at initiation of the new forks, but instead occurs at a time point coinciding with the end of SeqA-dependent origin sequestration. The finding suggests simultaneous origin de-sequestration and loss of SeqA from old replication forks.     

Pyrimidine dimers block simian virus 40 replication forks.

UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.     

Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro.

In discontinuous polyoma DNA replication, the synthesis of Okazaki fragments is primed by RNA. During viral DNA synthesis in nuclei isolated from infected cells, 40% of the nascent short DNA fragments had the polarity of the leading strand which, in theory, could have been synthesized by a continuous mechanism. To rule out that the leading strand fragments were generated by degradation of nascent DNA, they were further characterized. DNA fragments from a segment of the genome which replication forks pass in only one direction were strand separated. The sizes of the fragments from both strands were similar, suggesting that one strand was not specifically degraded. Most important, however, the majority of the Okazaki fragments of both strands were linked to RNA at their 5' ends. For identification, the RNA was labeled at the 5' ends by [beta-32P]GTP, internally by [3H]CTP, [3H]GTP, and [3H]UTP, or at the 3' ends by 32P transfer from adjacent [32P]dTMP residues. All three kinds of labeling indicated that an equal proportion of DNA fragments from the two strands was linked to RNA primers.     

Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae

Mrc1 associates with replication forks, where it transmits replication stress signals and is required for normal replisome pausing in response to nucleotide depletion. Mrc1 also plays a poorly understood role in DNA replication, which appears distinct from its role in checkpoint signaling. Here, we demonstrate that Mrc1 functions constitutively to promote normal replication fork progression. In mrc1Delta cells, replication forks proceed slowly throughout chromatin, rather than being specifically defective in pausing and progression through loci that impede fork progression. Analysis of genetic interactions with Rrm3, a DNA helicase required to resolve paused forks, indicates that Mrc1 checkpoint signaling is dispensable for the resolution of stalled replication forks and suggests that replication forks lacking Mrc1 create DNA damage that must be repaired by Rrm3. These findings elucidate a central role for Mrc1 in normal replisome function, which is distinct from its role as a checkpoint mediator, but nevertheless critical to genome stability.     

Bidirectional termination of chromosome replication in Escherichia coli

Summary Autoradiography was used to study the termination of replication of the circular chromosome of Escherichia coli. The experiments were conducted with cells in which termination occurred with a moderate amount of synchrony. Grain tracks were observed that demonstrated the approach at the replication terminus of the two replication forks involved in bidirectional replication. Other grain tracks were formed by replication forks that had met at the replication terminus. The frequency at which these patterns were observed indicates that most, if not all, terminations occur with both replication forks reaching the terminus at approximately the same time.     

Suppression of replication of SV40 and polyoma virus in mouse-human hybrids.

Mouse-human heterokaryons are permissive for the replication of both SV40 virus and polyoma virus. If the hybrids which develop from these heterokaryons segregate human chromosomes (mouse greater than human hybrids), the hybrids are permissive for replication of polyoma virus but not for replication of SV40 virus. If the subsequent hybrids segregate mouse chromosomes (human greater than mouse hybrids), such hybrids support the replication of SV40 virus but not the replication of polyoma virus, even when the hybrids contain at least one copy of each mouse chromosome. This indicates that during the transition from heterokaryon to hybrid cell, suppression of expression of species-specific function(s) required for the replication of these species-specific viruses occurs in parallel with the direction of chromosome loss and suppression of nucleolus organizer activity.     

Replication fork collapse at replication terminator sequences

Replication fork arrest is a source of genome re arrangements, and the recombinogenic properties of blocked forks are likely to depend on the cause of blockage. Here we study the fate of replication forks blocked at natural replication arrest sites. For this purpose, Escherichia coli replication terminator sequences Ter were placed at ectopic positions on the bacterial chromosome. The resulting strain requires recombinational repair for viability, but replication forks blocked at Ter are not broken. Linear DNA molecules are formed upon arrival of a second round of replication forks that copy the DNA strands of the first blocked forks to the end. A model that accounts for the requirement for homologous recombination for viability in spite of the lack of chromosome breakage is proposed. This work shows that natural and accidental replication arrests sites are processed differently.     

PARP is activated at stalled forks to mediate Mre11‐dependent replication restart and recombination

If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP‐ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea‐induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.     

Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins

The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Delta cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Delta dun1Delta cells and relieves the reliance on the DNA damage checkpoint pathway. Origin licensing and activation are controlled by the cyclin-Cdk complexes. Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.     

Exploring the roles of Mus81-Eme1/Mms4 at perturbed replication forks

Cells of all living organisms have evolved complex mechanisms that serve to stabilise, repair and restart stalled, blocked and broken replication forks. The heterodimeric Mus81-Eme1/Mms4 structure-specific endonuclease appears to play an important role(s) in homologous recombination-mediated processing of such perturbed forks. This enzyme has been implicated in the cleavage of stalled and blocked replication forks to initiate recombination, as well as in the processing of recombination intermediates that result from repairing damaged forks. In this review we assess the biochemical and genetic evidence for the mitotic role of Mus81-Eme1/Mms4 at replication forks and in repairing post-replication DNA damage. Mus81 appears to act when replication is impeded by genotoxins or by impairment of the replication machinery, or when arrested replication forks are not adequately protected. We discuss how its action is regulated by the S-phase cell cycle checkpoint, depending on the nature of the stalled or damaged fork. We also present a new way in which Mus81 may limit crossing over during the repair of post-replication gaps, and explore Mus81's interplay with other components of the recombination machinery, including the RecQ helicases that also play important roles in processing replication and recombination intermediates.     

Replication arrests during a single round of replication of the Escherichia coli chromosome in the absence of DnaC activity

We used a flow cytometric assay to determine the frequency of replication fork arrests during a round of chromosome replication in Escherichia coli. After synchronized initiation from oriC in a dnaC(Ts) strain, non-permissive conditions were imposed, such that active DnaC was not available during elongation. Under these conditions, about 18% of the cells failed to complete chromosome replication. The sites of replication arrests were random and occurred on either arm of the bidirectionally replicating chromosome, as stalled forks accumulated at the terminus from both directions. The forks at the terminal Ter sites disappeared in the absence of Tus protein, as the active forks could then pass through the terminus to reach the arrest site, and the unfinished rounds of replication would be completed without DnaC. In a dnaC2(Ts)rep double mutant, almost all cells failed to complete chromosome replication in the absence of DnaC activity. As inactivation of Rep helicase (the rep gene product) has been shown to cause frequent replication arrests inducing double-strand breaks (DSBs) in a replicating chromosome, DnaC activity appears to be essential for replication restart from DSBs during elongation.     

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G₁ phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.     

Replication fork reactivation in a dnaC2 mutant at non-permissive temperature in Escherichia coli

Replicative helicases unwind double-stranded DNA in front of the polymerase and ensure the processivity of DNA synthesis. In Escherichia coli, the helicase loader DnaC as well as factors involved in the formation of the open complex during the initiation of replication and primosomal proteins during the reactivation of arrested replication forks are required to recruit and deposit the replicative helicase onto single-stranded DNA prior to the formation of the replisome. dnaC2 is a thermosensitive allele of the gene specifying the helicase loader; at non-permissive temperature replication cannot initiate, but most ongoing rounds of replication continues through to completion (18% of dnaC2 cells fail to complete replication at non-permissive temperature). An assumption, which may be drawn from this observation, is that only a few replication forks are arrested under normal growth conditions. This assumption, however, is at odds with the severe and deleterious phenotypes associated with a null mutant of priA, the gene encoding a helicase implicated in the reactivation of arrested replication forks. We developed an assay that involves an abrupt inactivation of rounds of synchronized replication in a large population of cells, in order to evaluate the ability of dnaC2 cells to reactivate arrested replication forks at non-permissive temperature. We compared the rate at which arrested replication forks accumulated in dnaC2 priA(+) and dnaC2 priA2 cells and observed that this rate was lower in dnaC2 priA(+) cells. We conclude that while replication cannot initiate in a dnaC2 mutant at non-permissive temperature, a class of arrested replication forks (PriA-dependent and DnaC-independent) are reactivated within these cells.