Simian Virus 40 DNA Replication Research Articles

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-PKcs 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-PKcs 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.

A Wolf in Sheep's Clothing: SV40 Co-opts Host Genome Maintenance Proteins to Replicate Viral DNA

Simian virus 40 (SV40) was discovered in 1960 as a contaminant in early polio vaccines. Its discovery coincided with an explosion of knowledge in the new field of molecular biology, and SV40 was quickly adopted as a model to study eukaryotic genome structure, expression, replication, and cell growth regulation in cultured cells [1]. With a genome of only 5.2 kbp, SV40 relies heavily on host cell machinery to propagate, affording investigators a powerful tool to discover key host proteins that the virus manipulates. Indeed, a single multifunctional viral protein, the large tumor (T) antigen (Tag) (Figure 1A), is sufficient to orchestrate the replication of the viral mini-chromosome in infected monkey cells [2], [3]. The origin DNA binding domain of Tag binds specifically to the viral origin of DNA replication, and the C-terminal helicase domain of Tag unwinds parental DNA at SV40 replication forks. The development of a cell-free reaction containing purified Tag and primate cell extract enabled the identification of ten evolutionarily conserved host proteins that are necessary and sufficient, together with Tag, to replicate SV40 DNA in vitro [3], [4]. Figure 1 Assembly and activation of the SV40 pre-replication complex in vitro.

An Alternative Form of Replication Protein A Prevents Viral Replication in Vitro

Replication protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism. The "canonical" RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation. We demonstrate that the resulting "alternative" RPA (aRPA) complex has solution and DNA binding properties indistinguishable from the canonical RPA complex; however, aRPA is unable to support DNA replication and inhibits canonical RPA function. Two regions of RPA4, the putative L34 loop and the C terminus, are responsible for inhibiting SV40 DNA replication. Given that aRPA inhibits canonical RPA function in vitro and is found in nonproliferative tissues, these studies indicate that RPA4 expression may prevent cellular proliferation via replication inhibition while playing a role in maintaining the viability of quiescent cells.

Simian Virus 40 Large T Antigen Can Specifically Unwind the Central Palindrome at the Origin of DNA Replication

The hydrophilic channels between helicase domains of simian virus 40 (SV40) large T antigen play a critical role in DNA replication. Previous mutagenesis of residues in the channels identified one class of mutants (class A: D429A, N449S, and N515S) with normal DNA binding and ATPase and helicase activities but with a severely reduced ability to unwind origin DNA and to support SV40 DNA replication in vitro. Here, we further studied these mutants to gain insights into how T antigen unwinds the origin. We found that the mutants were compromised in melting the imperfect palindrome (EP) but normal in untwisting the AT-rich track. However, the mutants' defect in EP melting was not the major reason they failed to unwind the origin because supplying an EP region as a mismatched bubble, or deleting the EP region altogether, did not rescue their unwinding deficiency. These results suggested that specific separation of the central palindrome of the origin (site II) is an essential step in unwinding origin DNA by T antigen. In support of this, wild-type T antigen was able to specifically unwind a 31-bp DNA containing only site II in an ATPase-dependent reaction, whereas D429A and N515S failed to do so. By performing a systematic mutagenesis of 31-bp site II DNA, we identified discrete regions in each pentanucleotide necessary for normal origin unwinding. These data indicate that T antigen has a mechanism to specifically unwind the central palindrome. Various models are proposed to illustrate how T antigen could separate the central origin.

SV40 DNA replication: From the A gene to a nanomachine

Duplication of the simian virus 40 (SV40) genome is the best understood eukaryotic DNA replication process to date. Like most prokaryotic genomes, the SV40 genome is a circular duplex DNA organized in a single replicon. This small viral genome, its association with host histones in nucleosomes, and its dependence on the host cell milieu for replication factors and precursors led to its adoption as a simple and powerful model. The steps in replication, the viral initiator, the host proteins, and their mechanisms of action were initially defined using a cell-free SV40 replication reaction. Although our understanding of the vastly more complex host replication fork is advancing, no eukaryotic replisome has yet been reconstituted and the SV40 paradigm remains a point of reference. This article reviews some of the milestones in the development of this paradigm and speculates on its potential utility to address unsolved questions in eukaryotic genome maintenance.

Simian Virus 40 DNA Replication Is Dependent on an Interaction between Topoisomerase I and the C-Terminal End of T Antigen

Topoisomerase I (topo I) is needed for efficient initiation of simian virus 40 (SV40) DNA replication and for the formation of completed DNA molecules. Two distinct binding sites for topo I have been previously mapped to the N-terminal (residues 83 to 160) and C-terminal (residues 602 to 708) regions of T antigen. By mutational analysis, we identified a cluster of six residues on the surface of the helicase domain at the C-terminal binding site that are necessary for efficient binding to topo I in enzyme-linked immunosorbent assay and far-Western blot assays. Mutant T antigens with single substitutions of these residues were unable to participate normally in SV40 DNA replication. Some mutants were completely defective in supporting DNA replication, and replication was not enhanced in the presence of added topo I. The same mutants were the ones that were severely compromised in binding topo I. Other mutants demonstrated intermediate levels of activity in the DNA replication assay and were correspondingly only partially defective in binding topo I. Mutations of nearby residues outside this cluster had no effect on DNA replication or on the ability to bind topo I. These results strongly indicate that the association of topo I with these six residues in T antigen is essential for DNA replication. These residues are located on the back edges of the T-antigen double hexamer. We propose that topo I binds to one site on each hexamer to permit the initiation of SV40 DNA replication.

Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication

The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase alpha-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities, underlining the crucial role of specific protein-protein contacts during the entire initiation process.

Role of the Hydrophilic Channels of Simian Virus 40 T-Antigen Helicase in DNA Replication

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

Inhibition of Simian Virus 40 Large T Antigen Helicase Activity by Fluoroquinolones

Fluoroquinolones represent a potent group of antibiotics that inhibit bacterial DNA replication by targeting the essential bacterial enzymes gyrase and topoisomerase IV. Inhibition of gyrase activity by quinolones involves the interaction of these drugs with the helicase component of bacterial gyrase. DNA tumour viruses also encode helicases that are essential for their DNA replication in the host. In this study we have evaluated the effect of fluoroquinolones on viral DNA replication using the DNA tumour virus Simian virus 40 (SV40) as our model. Four different fluoroquinolones, namely, levofloxacin, trovafloxacin, ciprofloxacin and ofloxacin, were tested for their ability to inhibit viral DNA replication. We show here that all four quinolones tested were effective in the inhibition of SV40 plaque formation and DNA replication in CV1-P cells. In addition, we found that each of these quinolones was inhibitory to the helicase activity of SV40 large tumour antigen. Fluoroquinolones and their derivates may therefore be useful in the treatment and/or prevention of infection by SV40-homologous human DNA viruses that encode helicase activity for their survival.

Replication of simian virus 40 (SV40) DNA in virus-infected CV1 cells selectively permeabilized for small molecules by Staphylococcus aureus alpha-toxin: involvement of mitochondria in the fast O2-dependent regulation of SV40 DNA replication.

SV40 (simian virus 40)-infected CV1 cells were permeabilized with Staphylococcus aureus alpha-toxin for small molecules (<2 kDa) in a medium that supports DNA replication. Incorporation of [alpha-32P]dATP was shown to proceed at an essentially constant rate for at least 1 h. 32P-labelled DNA replication intermediates and products were analysed by alkaline sucrose density centrifugation. The results suggested that SV40 DNA replication in alpha-toxin-permeabilized CV1 cells occurred essentially as in vivo. After bromodeoxyuridine 5'-triphosphate-labelling and isopycnic banding, significant amounts of DNA density-labelled in both strands were detected from 110 min of permeabilization onwards, indicating repeated rounds of viral DNA replication in the permeabilized cells. Incubation of permeabilized SV40-infected cells under hypoxic culture conditions caused inhibition of SV40 DNA replication. As seen in unpermeabilized cells, SV40 DNA replication was inhibited at the stage of initiation. The inhibition of DNA replication induced by hypoxia was mimicked by AA (antimycin A), an inhibitor of mitochondrial respiration, and also by the replacement of glutamate, a substrate of mitochondrial respiration, by Hepes in the permeabilization medium. Inhibition of DNA replication was not mediated by intracellular ATP depletion. AA also inhibited SV40 DNA replication in unpermeabilized, normoxically incubated cells. Moreover, as in hypoxically incubated cells, the addition of glucose to SV40-infected cells incubated for several hours with AA induced a burst of new initiations followed by a nearly synchronous round of viral DNA replication. Taken together, these results indicate that mitochondria are involved in the oxygen-dependent regulation of SV40 DNA replication.

Coordinated Regulation of Replication Protein A Activities by Its Subunits p14 and p32

The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPADeltap14, consisting of p70 and p32. RPADeltap14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPADeltap14 complex p32 only minimally recognized the 3'-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPADeltap14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase alpha-primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on single-stranded DNA requires all three subunits to support DNA replication.

Relationship among location of T-antigen-induced DNA distortion, auxiliary sequences, and DNA replication efficiency.

T-antigen-induced DNA distortion was studied in a series of simian virus 40 (SV40) plasmid constructs whose relative replication efficiency ranges from 0.2 to 36. Bending was detected in the wild-type SV40 regulatory region consisting of three copies of the GC-rich 21-bp repeat but not in constructs with only one or two copies of the 21-bp repeat. In a construct with enhanced replication efficiency, bending occurred in a 69-bp cellular sequence located upstream of a single copy of the 21-bp repeat. Bending occurred both upstream of ori and in the three 21-bp repeats located downstream of ori in a construct with reduced replication efficiency. In a construct with no 21-bp repeats, DNA distortion occurred downstream of ori. The results indicate that SV40 DNA replication is enhanced when the structure of the regulatory region allows the DNA to form a bent structure upstream of the initial movement of the replication fork.

Regulation of Cellular and SV40 Virus Origins of Replication by Chk1-dependent Intrinsic and UVC Radiation-induced Checkpoints

DNA replication is inhibited by DNA damage through cis effects on replication fork progression and trans effects associated with checkpoints. In this study, we employed a combined pulse labeling and neutral-neutral two-dimensional gel-based approach to compare the effects of a DNA damaging agent frequently employed to invoke checkpoints, UVC radiation, on the replication of cellular and simian virus 40 (SV40) chromosomes in intact cells. UVC radiation induced similar inhibitory effects on the initiation and elongation phases of cellular and SV40 DNA replication. The initiation-inhibitory effects occurred independently of p53 and were abrogated by the ATM and ATR kinase inhibitor caffeine, or the Chk1 kinase inhibitor UCN-01. Inhibition of cellular origins was also abrogated by the expression of a dominant-negative Chk1 mutant. These results indicate that UVC induces a Chk1- and ATR or ATM-dependent checkpoint that targets both cellular and SV40 viral replication origins. Loss of Chk1 and ATR or ATM function also stimulated initiation of cellular and viral DNA replication in the absence of UVC radiation, revealing the existence of a novel intrinsic checkpoint that targets both cellular and SV40 viral origins of replication in the absence of DNA damage or stalled DNA replication forks. This checkpoint inhibits the replication in early S phase cells of a region of the repetitive rDNA locus that replicates in late S phase. The ability to detect these checkpoints using the well characterized SV40 model system should facilitate analysis of the molecular basis for these effects.

Oxygen-dependent Regulation of in VivoReplication 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.

Different regions of primase subunit p48 control mouse polyomavirus and simian virus 40 DNA replication in vitro.

DNA polymerase alpha-primase (pol-prim), a complex consisting of four subunits, is the major species-specific factor for mouse polyomavirus (PyV) and simian virus 40 (SV40) DNA replication. Although p48 is the most conserved subunit of pol-prim, it is required for in vitro PyV DNA replication but can inhibit cell-free SV40 DNA replication. Production of chimeric human-mouse p48 revealed that different regions of p48 are involved in supporting PyV DNA replication and inhibiting SV40 DNA replication. The N and C-terminal parts of p48 do not have species-specific functions in cell-free PyV DNA replication, but the central part (amino acids [aa] 129 to 320) controls PyV DNA replication in vitro. However, PyV T antigen physically binds to mouse, human, and chimeric pol-prim complexes independently, whether they support PyV DNA replication or not. In contrast to the PyV system, the inhibitory effects of mouse p48 on SV40 DNA replication are mediated by N- and C-terminal regions of p48. Thus, a chimeric p48 containing human aa 1 to 128, mouse aa 129 to 320, and human aa 321 to 418 is active in both PyV and SV40 DNA replication in vitro.

Regulation of DNA Replication after Heat Shock by Replication Protein A-Nucleolin Interactions

Heat shock inhibits replicative DNA synthesis, but the underlying mechanism remains unknown. We investigated mechanistic aspects of this regulation in melanoma cells using a simian virus 40 (SV40)-based in vitro DNA replication assay. Heat shock (44 degrees C) caused a monotonic inhibition of cellular DNA replication following exposures for 5-90 min. SV40 DNA replication activity in extracts of similarly heated cells also decreased after 5-30 min of exposure, but returned to near control levels after 60-90 min of exposure. This transient inhibition of SV40 DNA replication was eliminated by recombinant replication protein A (rRPA), suggesting a regulatory process targeting this key DNA replication factor. SV40 DNA replication inhibition was associated with a transient increase in the interaction between nucleolin and RPA that peaked at 20-30 min. Because binding to nucleolin compromises the ability of RPA to support SV40 DNA replication, we suggest that the observed interaction reflects a mechanism whereby DNA replication is regulated after heat shock. The relevance of this interaction to the regulation of cellular DNA replication is indicated by the transient translocation in heated cells of nucleolin from the nucleolus into the nucleoplasm with kinetics very similar to those of SV40 DNA replication inhibition and of RPA-nucleolin interaction. Because the targeting of RPA by nucleolin in heated cells occurs in an environment that preserves the activity of several essential DNA replication factors, active processes may contribute to DNA replication inhibition to a larger degree than presently thought. RPA-nucleolin interactions may reflect an early step in the regulation of DNA replication, as nucleolin relocalized into the nucleolus 1-2 h after heat exposure but cellular DNA replication remained inhibited for up to 8 h. We propose that the nucleolus functions as a heat sensor that uses nucleolin as a signaling molecule to initiate inhibitory responses equivalent to a checkpoint.

Bleomycin-induced alterations in DNA replication: relationship to DNA damage.

Bleomycin (BLM), a well-known DNA scission agent, is assumed to inhibit intracellular DNA replication by damaging the DNA template (cis-acting mechanism), although other DNA damaging compounds can alter DNA replication through modulation of crucial replication factor(s) (trans-acting mechanism). The present study examines the relationship between DNA damage and inhibition of replication caused by BLM in the well-defined simian virus 40 (SV40) intracellular and cell-free in vitro systems. Treatment of SV40-infected BSC-1 cells for 2 h with BLM at 50 microg/mL, induced 0.3 break/viral genome. Under the same treatment conditions, analysis of replication intermediates on two-dimensional gels showed a decrease in both mass of SV40 replication intermediates and replication activity. The mass of SV40 intermediates was decreased to about 30%, whereas replication activity was reduced to less than 5%. These results suggest that BLM inhibits both initiation and elongation phases of SV40 replication. In a cell-free DNA replication system, extracts from BLM-treated cells (50 micro/mL) were able to support SV40 DNA replication by only 50%. In this study, non-drug-treated DNA template was used, implying that BLM can induce a trans-acting effect. Finally, the drug-induced effects on SV40 DNA replication in cell-free and intracellular viral systems were compared to the effects on genomic DNA replication in BSC-1 cells. Overall, the results support the concept that BLM-induced inhibition of DNA replication occurs by both trans- (inhibition of replication of nondamaged template) and cis-acting mechanisms (template damage).

Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis.

Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication. Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication. To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S.pombe subunits and compared these preparations for various enzymatic activities. Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All heterologous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol) alpha and delta. A strong correlation between SV40 DNA replication activity and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol alpha-primase complex was observed among the heterologous RPAs. Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis. In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.

Structure of Simian Virus 40 DNA Replicated by Herpes Simplex Virus Type 1

Replicating herpes simplex virus type 1 (HSV-1) DNA is known to form large branched structures. The aim of this study was to define whether HSV-1-specific DNA elements in cis play a critical role in formation of this structure. We did this by investigating the structure of heterologous simian virus 40 (SV40) DNA, which is replicated in HSV-infected cells by SV40 large T-antigen and defined HSV-encoded replication factors (e.g., DNA polymerase, single-stranded DNA-binding protein, and helicase-primase). During this process, extrachromosomal concatemeric DNA replication products are formed, indicating a herpesvirus-specific replication mode. In this study, we found that the replicating SV40 DNA consisted of a complex branched structure indistinguishable from that of replicating HSV DNA. Thus, no HSV-specific DNA element is necessary in cis for the formation of the large branched structure during HSV DNA replication. The trans-acting HSV DNA replication proteins seem to be sufficient to generate these complex structures. Moreover, replicating SV40 DNA showed a high frequency of homologous recombination events, which is typical for HSV DNA replication. However, in contrast to HSV origin-bearing amplicon plasmids, SV40 plasmids bearing the HSV cleavage-packaging signal were not efficiently processed to linear 150-kb DNA packaged into HSV capsids. This indicates that initiation of DNA synthesis on HSV-ori determines some, yet undefined, property of replicating HSV DNA, which is crucial for regular processing of the replication intermediates to daughter genomes.

Phosphorylation of simian virus 40 T antigen on Thr 124 selectively promotes double-hexamer formation on subfragments of the viral core origin.

Cell cycle-dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DNA replication. A T-ag molecule containing a Thr-->Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but to be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of the T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T124A molecules on the SV40 origin. Herein it is demonstrated that hexamer formation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124A molecules are inhibitors of T-ag double hexamer formation. These and related studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV40 DNA replication are discussed.