Interactions between DNA helicases and frozen topoisomerase IV-quinolone-DNA ternary complexes.

Collisions between replication forks and topoisomerase-drug-DNA ternary complexes result in the inhibition of DNA replication and the conversion of the normally reversible ternary complex to a nonreversible form. Ultimately, this can lead to the double strand break formation and subsequent cell death. To understand the molecular mechanisms of replication fork arrest by the ternary complexes, we have investigated molecular events during collisions between DNA helicases and topoisomerase-DNA complexes. A strand displacement assay was employed to assess the effect of topoisomerase IV (Topo IV)-norfloxacin-DNA ternary complexes on the DnaB, T7 gene 4 protein, SV40 T-antigen, and UvrD DNA helicases. The ternary complexes inhibited the strand displacement activities of these DNA helicases. Unlike replication fork arrest, however, this general inhibition of DNA helicases by Topo IV-norfloxacin-DNA ternary complexes did not require the cleavage and reunion activity of Topo IV. We also examined the reversibility of the ternary complexes after collisions with these DNA helicases. UvrD converted the ternary complex to a nonreversible form, whereas DnaB, T7 gene 4 protein, and SV40 T-antigen did not. These results suggest that the inhibition of DnaB translocation may be sufficient to arrest the replication fork progression but it is not sufficient to generate cytotoxic DNA lesion.     

Dimerization of simian virus 40 T-antigen hexamers activates T-antigen DNA helicase activity.

Chromosomal DNA replication in higher eukaryotes takes place in DNA synthesis factories containing numerous replication forks. We explored the role of replication fork aggregation in vitro, using as a model the simian virus 40 (SV40) large tumor antigen (T antigen), essential for its DNA helicase and origin-binding activities. Previous studies have shown that T antigen binds model DNA replication forks primarily as a hexamer (TAgH) and to a lesser extent as a double hexamer (TAgDH). We find that DNA unwinding in the presence of ATP or other nucleotides strongly correlates with the formation of TAgDH-DNA fork complexes. TAgH- and TAgDH-fork complexes were isolated, and the TAgDH-bound fork was denatured at a 15-fold-higher rate during the initial times of unwinding. TAgDH bound preferentially to a DNA substrate containing a 50-nucleotide bubble, indicating the bridging of each single-stranded DNA/duplex DNA junction, and this DNA molecule was also unwound at a high rate. Both the TAgH- and TAgDH-fork complexes were relatively stable, with the half-life of the TAgDH-fork complex greater than 40 min. Our data therefore indicate that the linking of two viral replication forks serves to activate DNA replication.     

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PK_cs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PK_cs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication.     

UV lesions located on the leading strand inhibit DNA replication but do not inhibit SV40 T-antigen helicase activity

DNA replication in eucaryotic cells involves a variety of proteins which synthesize the leading and lagging strands in an asymmetric coordinated manner. To analyse the effect of this asymmetry on the translesion synthesis of UV-induced lesions, we have incubated SV40 origin-containing plasmids with a unique site-specific cis, syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct on either the leading or lagging strand template with DNA replication-competent extracts made from human HeLa cells. Two dimensional agarose gel electrophoresis analyses revealed a strong blockage of fork progression only when the UV lesion is located on the leading strand template. Because DNA helicases are responsible for unwinding duplex DNA ahead of the fork and are then the first component to encounter any potential lesion, we tested the effect of these single photoproducts on the unwinding activity of the SV40 T antigen, the major helicase in our in vitro replication assay. We showed that the activity of the SV40 T-antigen helicase is not inhibited by UV-induced DNA lesions in double-stranded DNA substrate.     

Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.

DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.     

Inactivation of topoisomerase I or II may lead to recombination or to aberrant replication termination on both SV40 and yeast 2 μm DNA

Topoisomerase I is believed to be sufficient for early replication of circular viral genomes such as those of SV40 and of yeast plasmids. Topoisomerase II is required for the decatenation of the daughter genomes and probably also for fork elongation during the later stages of SV40 replication. Using the neutral-neutral two-dimensional gel system, we have followed the progression of replication of both SV40 and the yeast 2μm plasmid under various conditions of topoisomerase inhibition. During SV40 replication, inhibition of topoisomerase II by VP16, VM26 or hypertonic shock (but not by merbarone), and inhibition of topoisomerase I by camptothecin all led to the accumulation of aberrant DNA structures containing two almost completely replicated genomes. These aberrant structures resembled either recombination intermediates or late Cairns structures in which the site of replication termination had shifted and now mapped to a continuum of sites throughout the genome. Replication of the 2 μm plasmid in a topoisomerase II- but not a topoisomerase I-deficient yeast gave rise to very similar structures. The data suggest that inactivation of topoisomerase I or II either stimulates recombination or, by differentially affecting replication fork progression, leads to aberrant replication termination. Edited by: J. Huberman     

A dual role of BRCA1 in two distinct homologous recombination mediated repair in response to replication arrest

Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency.     

The fate of parental nucleosomes during SV40 DNA replication.

The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.     

Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells

Exposure of eukaryotic cells to ultraviolet light results in a temporary inhibition of DNA replication as well as a temporary blockage of DNA fork progression. Recently there has been considerable debate as to whether the (5-6)cyclobutane pyrimidine dimer, the pyrimidine(6-4)pyrimidone lesion or both are responsible for these effects. Using cell lines that repair both of these lesions (CHO AA8), only (6-4) lesions (CHO UV61) or neither (CHO UV5), we have shown that in rodent cells both lesions appear to play a role in both the inhibition of thymidine incorporation and the blockage of DNA fork progression. Specifically, after exposure to 2.5 J/m2, AA8 cells recover normal rates of DNA replication within 5 h after exposure, while UV5 cells exhibit a greater depression in thymidine incorporation for at least 10 h. UV61 cells, on the other hand, show an intermediate response, both with respect to the extent of the initial depression and the rate of recovery of thymidine incorporation. UV61 cells also exhibit an intermediate response with respect to blockage of DNA fork progression. In previous publications we have shown that UV5 cells exhibit extensive blockage of DNA fork progression and only limited recovery of this effect within the first 5 h after exposure to UV. In this report we show that UV61 cells exhibit a more extensive blockage of fork progression than is observed in AA8 cells. These blocks also appear to be removed (or overcome) more slowly than in the AA8 cells, but more rapidly than in UV5 cells. Taken together we conclude that both lesions appear to be involved in the initial depression in thymidine incorporation and the initial blockage of DNA fork progression in rodent cells. These data also indicate that (6-4) lesions may be responsible for the prolonged depression in thymidine incorporation and the prolonged blockage of DNA fork progression observed in UV5 cells.     

Oxygen-dependent regulation of in vivo replication of simian virus 40 DNA is modulated by glucose

Simian virus 40 (SV40)-infected CV1 cells exposed to hypoxia show an inhibition of viral replication. Reoxygenation after several hours of hypoxia results in new initiations followed by a nearly synchronous round of SV40 replication. In this communication, we examined the effect of glucose on inhibition of viral DNA replication under hypoxia. We found that glucose stimulated SV40 DNA replication under hypoxia in two different ways. First, the rate of DNA synthesis, i.e. the fork propagation rate, increased. This effect seemed to be mediated by inhibition of mitochondrial respiration by glucose (Crabtree effect). Inhibition of mitochondrial respiration probably resulted in a higher intracellular oxygen concentration and an activation of oxygen-dependent ribonucleotide reductase, which provides the precursors for DNA synthesis. This glucose effect was consequently strongly dependent on the strength of hypoxia and the extent of intracellular respiration; hypoxic gassing with 10 ppm instead of 200-400 ppm O(2) or treatment of hypoxic cells with a mitochondrial uncoupler (carbonyl cyanide m-chlorophenylhydrazone) reduced the glucose effect on replication, whereas antimycin A, an inhibitor of respiration, increased it. The second effect of glucose concerned initiation, i.e. stimulation of unwinding of the viral origin. This effect was not influenced by the strength of hypoxia or the extent of cellular respiration and seemed, therefore, not to be mediated through a Crabtree effect. No evidence for a direct correlation between the cellular ATP concentration and the extent of SV40 replication under hypoxia was found. The effect of glucose on replication under hypoxia was not restricted to SV40-infected CV1 cells but was also detectable in HeLa cells. This suggests it to be a mechanism of more general validity.     

Pausing of simian virus 40 DNA replication fork movement in vivo by (dG-dA)n·(dT-dC)n tracts

We have earlier demonstrated that a sequence bordering an amplified DNA segment and containing the unusual sequence (dG-dA)_n·(dT-dC)_n could slow replication fork movement [Rao et al., Nucleic Acids Res. 16 (1988) 8077–8094]. This was done by cloning the unusual sequence in simian virus 40 (SV40) and following the rate of incorporation of radioactively labeled nucleotides into various regions of the SV40 genome. In the present study, we have analyzed the in vivo replicative intermediates of the SV40 variants containing the unusual sequences by a two-dimensional gel electrophoretic technique. We found that the technique can be used to detect minor pauses in DNA replication and demonstrated that the cloned (dG-dA)_n·(dT-dC)_n tracts, that can potentially adopt triplex structures, could slow DNA replication fork movement. A sequence from the plasmid pUC18 did not slow fork movement when cloned in the same locus of SV40. The pause caused by the alternating guanosine-adenosine repeats might play a role in the regulation of DNA replication and gene amplification in vivo.     

Aphidicolin-induced topological and recombinational events in simian virus 40.

Highly compacted (40S) SV40 DNA replication intermediates formed in vivo during aphidicolin exposure and immediately broke down in two stages. In the rapid initial stage, single strand DNA breaks caused loss of superhelicity in the 40S replication intermediates. This DNA breakage was accompanied by the formation of strong, permanent protein-DNA crosslinks which reached a maximum as nicking of the aberrant DNA replication intermediates was completed. These protein-associated DNA strand breaks were not repaired. In the slower second stage of breakdown, the aberrant DNA replication intermediates remained nicked and strongly associated with protein as they underwent DNA replication fork breakage and recombinational changes to produce high molecular weight forms.     

A novel assay for examining the molecular reactions at the eukaryotic replication fork: activities of replication protein A required during elongation.

Studies to elucidate the reactions that occur at the eukaryotic replication fork have been limited by the model systems available. We have established a method for isolating and characterizing Simian Virus 40 (SV40) replication complexes. SV40 rolling circle complexes are isolated using paramagnetic beads and then incubated under replication conditions to obtain continued elongation. In rolling circle replication, the normal mechanism for termination of SV40 replication does not occur and the elongation phase of replication is prolonged. Thus, using this assay system, elongation phase reactions can be examined in the absence of initiation or termination. We show that the protein requirements for elongation of SV40 rolling circles are equivalent to complete SV40 replication reactions. The DNA produced by SV40 rolling circles is double-stranded, unmethylated and with a much longer length than the template DNA. These properties are similar to those of physiological replication forks. We show that proteins associated with the isolated rolling circles, including SV40 T antigen, DNA polymerase alpha, replication protein A (RPA) and RF-C, are necessary for continued DNA synthesis. PCNA is also required but is not associated with the isolated complexes. We present evidence suggesting that synthesis of the leading and lagging strands are co-ordinated in SV40 rolling circle replication. We have used this system to show that both RPA-protein and RPA-DNA interactions are important for RPA's function in elongation.     

Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures

The structure of replicating simian virus 40 (SV40) minichromosomes was studied by DNA crosslinking with trimethyl-psoralen. The procedure was used both in vitro with extracted SV40 minichromosomes as well as in vivo with SV40-infected cells. Both procedures gave essentially the same results. Mature SV40 minichromosomes are estimated to contain about 27 nucleosomes (error +/- 2), except for those molecules with a nucleosome-free gap, which are interpreted to contain 25 nucleosomes (error +/- 2). In replicative intermediates, nucleosomes are present in the unreplicated parental stem with the replication fork possibly penetrating into the nucleosomal DNA before the histone octamer is removed. Nucleosomes reassociate on the newly replicated DNA branches at distances from the branch point of 225 ( +/- 145) nucleotides on the leading strand and of 285( +/- 120) nucleotides on the lagging strand. In the presence of cycloheximide, daughter duplexes contained unequal numbers of nucleosomes, supporting dispersive and random segregation of parental nucleosomes. These were arranged in clusters with normal nucleosome spacing. We detected a novel type of interlocked dimer comprising two fully replicated molecules connected by a single-stranded DNA bridge. We cannot decide whether these dimers represent hemicatenanes or whether the two circles are joined by a Holliday-type structure. The joining site maps within the replication terminus. We propose that these dimers represent molecules engaged in strand segregation.     

Inhibition of initiation of simian virus 40 DNA replication during acute response of cells irradiated by ultraviolet light.

To study the mechanism by which ultraviolet (UV) light inhibits DNA replication, we examined the effects of UV 254 nm irradiation on the replication of simian virus 40 (SV40) DNA and SV40-based plasmid in monkey cells. The study was designed to determine the relative contributions made by inhibition of replication initiation and chain elongation to the immediate inhibition of DNA replication following UV irradiation. We used two-dimensional neutral-alkaline electrophoresis to examine the behaviour of replication intermediates unambiguously. Kinetic analysis using this technique showed that initiation of replication started to decline at 15 min post-irradiation. When the pulse label incorporated in SV40 replication intermediates before irradiation was chased for 1 h, most of the label was found in mature Form I and II molecules. This indicated that replication elongation took place on damaged template. We also used a transfection technique to show that heavily irradiated plasmids replicated efficiently in unirradiated transfected cells. By the transfection technique, we observed that UV irradiation of host cells dose-dependently inhibited replication of transfected non-irradiated plasmids, suggesting that the inhibition of DNA replication is due to a global change in cellular physiology induced by UV. This change was also apparent from poor staining of the chromatin by fluorescent-DNA-binding dyes immediately after UV irradiation of intact cells. We conclude that a significant fraction of chain elongation proceeds on damaged templates and DNA replication during the acute response of cells irradiated with UV is mainly controlled by the inhibition of replication initiation.     

Roles of replication protein A and DNA-dependent protein kinase in the regulation of DNA replication following DNA damage.

Exposure of mammalian cells to DNA damage-inducing agents (DDIA) inhibits ongoing DNA replication. The molecular mechanism of this inhibition remains to be elucidated. We employed a simian virus 40 (SV40) based in vitro DNA replication assay to study biochemical aspects of this inhibition. We report here that the reduced DNA replication activity in extracts of DDIA-treated cells is partly caused by a reduction in the amount of replication protein A (RPA). We also report that the dominant inhibitory effect is caused by the DNA-dependent protein kinase (DNA-PK) which inactivates SV40 T antigen (TAg) by phosphorylation. The results demonstrate that RPA and DNA-PK are involved in the regulation of viral DNA replication after DNA damage and suggest that analogous processes regulate cellular DNA replication with the DNA-PK targeting the functional homologues of TAg.     

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.     

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.