Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans.

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.

Lagging Strand Initiation Processes in DNA Replication of Eukaryotes-Strings of Highly Coordinated Reactions Governed by Multiprotein Complexes.

In their influential reviews, Hanahan and Weinberg coined the term 'Hallmarks of Cancer' and described genome instability as a property of cells enabling cancer development. Accurate DNA replication of genomes is central to diminishing genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase (Pol-prim), during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exonucleases during double-strand break repair are discussed.

SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation.

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that while Tag131-627 (V350E/P417D) and Tag131-627 (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag1-708 and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.

Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities.

Polyomavirus infections occur commonly in humans and are normally nonfatal. However, in immunocompromised individuals, they are intractable and frequently fatal. Due to a lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections, has been repurposed as an antipolyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as a broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as antipolyomavirus agents are still under research. Here, polyomavirus large tumor antigen (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system utilizing only purified proteins. We present evidence that DNA helicase and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl ester derivatives of cidofovir, suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) binding activities of Tags. These findings show that the alkoxyalkyl ester derivatives of cidofovir are effective in vitro without undergoing further conversions, and we conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.

ATPase activity of human binding immunoglobulin protein (BiP) variants is enhanced by signal sequence and physiological concentrations of Mn2.

B-cell immunoglobulin binding protein (BiP) is an essential endoplasmic reticulum (ER) chaperone normally found in the ER lumen. However, BiP also has other extracellular and intracellular functions. As it is unclear whether peripheral BiP has a signal and/or ER retention sequence, here we produced and biochemically characterised four variants of BiP. The variants differed depending on the presence or the absence of signal and ER retention peptides. Proteins were purified using nickel affinity chromatography, and variant size and quality were confirmed using SDS/PAGE gels. The thermal denaturing temperature of these proteins was found to be 46-47 °C. In addition, we characterised nucleotide binding properties in the absence and the presence of divalent cations. Interestingly, in the absence of cations, ADP has a higher binding affinity to BiP than ATP. The presence of divalent cations results in a decrease of the Kd values of both ADP and ATP, indicating higher affinities of both nucleotides for BiP. ATPase assays were carried out to study the enzyme activity of these variants and to characterise the kinetic parameters of BiP variants. Variants with the signal sequence had higher specific activities than those without. Both Mg2+ and Mn2+ efficiently stimulated the ATPase activity of these variants at low micromolar concentrations, whereas calcium failed to stimulate BiP ATPase. Our novel findings indicate the potential functionality of BiP variants that retain a signal sequence, and also reveal the effect of physiological concentrations of cations on the nucleotide binding properties and enzyme activities of all variants.

Interplay of DNA damage and cell cycle signaling at the level of human replication protein A.

Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in human cell cycle and DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-strand break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phases of the cell cycle. All three subunits of RPA contain phosphorylation sites but the exact set of HR-relevant phosphorylation sites on RPA is unknown. In this study, a high resolution capillary isoelectric focusing immunoassay, used under native conditions, revealed the isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Moreover, the phosphorylation sites of chromatin-bound and cytosolic RPA in S and G2 phases were identified by western and IEF analysis with all available phosphospecific antibodies for RPA2. Strikingly, most of the RPA heterotrimers in control G2 cells are phosphorylated with 5 isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33. DNA damaged cells in G2 had 9 isoforms with up to 14 phosphates. DNA damage isoforms contained pSer4/8, pSer12, pThr21, pSer23, and pSer33 on RPA2 and up to 8 unidentified phosphorylation sites.

PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress.

Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation.

Basic fibroblast growth factor modifies the hypoxic response of human bone marrow stromal cells by ERK-mediated enhancement of HIF-1α activity.

Human bone marrow stromal cells (hBMSCs, also known as bone marrow-derived mesenchymal stem cells) are promising tools for the cellular therapy of human pathologies related to various forms of hypoxia. Although the current concepts of their clinical use include the expansion of hBMSC in standard cell culture conditions, the effect of the mitogen-driven ex vivo expansion on the adaptation to the hypoxic environment is unknown. Here, we provide data that the basic fibroblast growth factor (FGF2) enhances the induction of a wide range of hypoxia-related adaptive genes in hypoxic hBMSCs. We identified that the FGF2 signal is transmitted by the ERK pathway similar to that of hypoxia that also utilises the distal elements of the same signalling machinery including the extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase kinases (MEK1/2) in hBMSCs. We found that the simultaneous activation of ERK1/2 by FGF2 and hypoxia transforms the activation dynamics from oscillatory into sustained one. Activated ERKs co-localise with stabilised hypoxia inducible factor-1α (HIF-1α) followed by the reduction of its nuclear mobility as well as increased DNA binding capacity leading to the up-regulation of hypoxia-adaptive genes. Our findings indicate that the status of the ERK pathway has significant impacts on the molecular adaptation of hBMSCs to the hypoxic milieu.

Stimulation of BK virus DNA replication by NFI family transcription factors.

BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.

Inhibition of human BK polyomavirus replication by small noncoding RNAs.

Small noncoding RNAs regulate a variety of cellular processes, including genomic imprinting, chromatin remodeling, replication, transcription, and translation. Here, we report small replication-regulating RNAs (srRNAs) that specifically inhibit DNA replication of the human BK polyomavirus (BKV) in vitro and in vivo. srRNAs from FM3A murine mammary tumor cells were enriched by DNA replication assay-guided fractionation and hybridization to the BKV noncoding control region (NCCR) and synthesized as cDNAs. Selective mutagenesis of the cDNA sequences and their putative targets suggests that the inhibition of BKV DNA replication is mediated by srRNAs binding to the viral NCCR, hindering early steps in the initiation of DNA replication. Ectopic expression of srRNAs in human cells inhibited BKV DNA replication in vivo. Additional srRNAs were designed and synthesized that specifically inhibit simian virus 40 (SV40) DNA replication in vitro. These observations point to novel mechanisms for regulating DNA replication and suggest the design of synthetic agents for inhibiting replication of polyomaviruses and possibly other viruses.

Host-specific replication of BK virus DNA in mouse cell extracts is independently controlled by DNA polymerase alpha-primase and inhibitory activities.

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase alpha-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).

Eukaryotic single-stranded DNA binding proteins: central factors in genome stability.

The single-stranded DNA binding proteins (SSBs) are required to maintain the integrity of the genome in all organisms. Replication protein A (RPA) is a nuclear SSB protein found in all eukaryotes and is required for multiple processes in DNA metabolism such as DNA replication, DNA repair, DNA recombination, telomere maintenance and DNA damage signalling. RPA is a heterotrimeric complex, binds ssDNA with high affinity, and interacts specifically with multiple proteins to fulfil its function in eukaryotes. RPA is phosphorylated in a cell cycle and DNA damage-dependent manner with evidence suggesting that phosphorylation has an important function in modulating the cellular DNA damage response. Considering the DNA-binding properties of RPA a mechanism of "molecular counting" to initiate DNA damage-dependent signalling is discussed. Recently a human homologue to the RPA2 subunit, called RPA4, was discovered and RPA4 can substitute for RPA2 in the RPA complex resulting in an "alternative" RPA (aRPA), which can bind to ssDNA with similar affinity as canonical RPA. Additional human SSBs, hSSB1 and hSSB2, were recently identified, with hSSB1 being localized in the nucleus and having implications in DNA repair. Mitochondrial SSBs (mtSSBs) have been found in all eukaryotes studied. mtSSBs are related to prokaryotic SSBs and essential to main the genome stability in eukaryotic mitochondria. Recently human mtSSB was identified as a novel binding partner of p53 and that it is able to stimulate the intrinsic exonuclease activity of p53. These findings and recent results associated with mutations in RPA suggest a link of SSBs to cancer.

The use of transgenic mice in cancer and genome stability research.

The development of effective cancer therapeutics is an important goal of modern biomedical sciences. To identify potential cancer therapeutic targets, the processes involved in tumorigenesis must be understood at all levels, which requires the development of model systems accurately mimicing tumor development. Cancer is the general name given to a variety of complex diseases characterised by uncontrolled cell proliferation. Cancer development is dependent not only on the changes occurring within the transformed cells, but also on the interactions of the cells with their microenvironment. The majority of our current understanding of carcinogenesis comes from the in vitro analysis of late-stage tumor tissue removed from cancer patients. While this has elucidated many genomic changes experienced by cancer cells, it provides little information about the factors influencing early-stage cancer development in vivo. Also certain hallmarks of cancer, such as metastasis and angiogenesis, are impossible to study in vitro. The mouse has become an important model for studying the in vivo aspects of human cancer development. Transgenic mouse models have been engineered to develop cancers, which accurately mimic their human counterparts, and have potential applications to test the effectiveness of novel cancer therapeutics. One of the most promising transgenic mouse models of human cancer arises from mice engineered with genomic instability. These transgenic models have been shown to develop human-like cancers and have the potential to provide insights into the molecular events occurring in earliest stages of tumorigenesis in vivo.

Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis.

The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S- and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNA-PK, and is associated with activation of the Chk2 pathway.

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication.

Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase alpha-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein-protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end.

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.

Comparison of DNA replication in Xenopus laevis and Simian Virus 40.

DNA replication is a fundamental process within the cell cycle. The exact duplication of the genetic information ensures genome stability. Extensive research has identified the principal players required for the sequential processes: origin-licensing (a controlled order of events giving a chromosome site the potential to be initiated within the S phase of the same cell cycle); initiation (by removing the license a previous licensed site is transformed into a site where the DNA helix starts to melt); and DNA replication (copying the parental DNA by leading and lagging strand DNA-synthesis). The present report compares the advantages and limitations of studying DNA replication in the model systems Xenopus laevis (X. laevis) and in Simian Virus 40 (SV40).

The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism.

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the beta-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the beta-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.

DNA polymerase epsilon associates with the elongating form of RNA polymerase II and nascent transcripts.

DNA polymerase epsilon co-operates with polymerases alpha and delta in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase epsilon and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase epsilon was also shown to UV crosslink specifically alpha-amanitin-sensitive transcripts, unlike DNA polymerase alpha that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase epsilon, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase epsilon in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase epsilon complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.

Partial proteolysis of simian virus 40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly.

Simian virus 40 large tumor antigen (Tag) is a multi-functional viral protein that binds specifically to SV40 origin DNA, serves as the replicative DNA helicase, and orchestrates the assembly and operation of the viral replisome. Tag associated with Mg-ATP forms hexamers and, in the presence of SV40 origin DNA, double hexamers. Limited tryptic digestion of monomeric Tag revealed three major stable structural domains. The N-terminal domain spans amino acids 1-130, the central domain comprises amino acids 131-476, and the C-terminal domain extends from amino acid 513 to amino acid 698. Co-immunoprecipitation of digestion products of monomeric Tag suggests that the N-terminal domain associates stably with sequences located in the central region of the same Tag molecule. Hexamer formation protected the tryptic cleavage sites in the exposed region between the central and C-terminal domains. Upon hexamerization, this exposed region also became less accessible to a monoclonal antibody whose epitope maps in that region. The tryptic digestion products of the soluble hexamer and the DNA-bound double hexamer were indistinguishable. A low-resolution model of the intramolecular and intermolecular interactions among Tag domains in the double hexamer is proposed.