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The 5.5 protein (T7p32) of coliphage T7 (5.5 T7 ) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5 T7 protein is insoluble when expressed in Escherichia coli , but we find that 5.5 T7 can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5 T7 binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5 T7 can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se . Homologues of the 5.5 T7 protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5 T7 protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression.

Summary Proper coordination of DNA replication with cell growth and division is critical for production of viable progeny. In bacteria, coordination of DNA replication with cell growth is generally achieved by controlling activity of the replication initiator DnaA and its access to the chromosomal origin of replication, oriC. Here we describe a previously unknown mechanism for regulation of DnaA. YabA, a negative regulator of replication initiation in Bacillus subtilis, interacts with DnaA and DnaN, the sliding (processivity) clamp of DNA polymerase. We found that in vivo, YabA associated with the oriC region in a DnaA-dependent manner and limited the amount of DnaA at oriC. In vitro, purified YabA altered binding of DnaA to DNA by inhibiting cooperativity. Although previously undescribed, proteins that directly inhibit cooperativity may be a common mechanism for regulating replication initiation. Conditions that cause release of DnaN from the replisome, or overproduction of DnaN, caused decreased association of YabA and increased association of DnaA with oriC. This effect of DnaN, either directly or indirectly, is likely responsible, in part, for enabling initiation of a new round of replication following completion of a previous round.

The onset of chromosomal DNA replication requires highly precise and reproducible interactions between initiator proteins and replication origins to assemble a pre-replicative complex (pre-RC) that unwinds the DNA duplex. In bacteria, initiator protein DnaA, bound to specific high- and low-affinity recognition sites within the unique oriC locus, comprises the pre-RC, but how complex assembly is choreographed to ensure precise initiation timing during the cell cycle is not well understood. In this study, we present evidence that higher-order DnaA structures are formed at oriC when DnaA monomers are closely positioned on the same face of the DNA helix by interaction with two oppositely oriented essential arrays of closely spaced low-affinity DnaA binding sites. As DnaA levels increase, peripheral high-affinity anchor sites begin cooperative loading of the arrays, which is extended by sequential binding of additional DnaA monomers resulting in growth of the complexes towards the centre of oriC. We suggest that this polarized assembly of unique DnaA oligomers within oriC plays an important role in mediating pre-RC activity and may be a feature found in all bacterial replication origins.

Mgs1, the budding yeast homolog of mammalian Werner helicase-interacting protein 1 (WRNIP1/WHIP), contributes to genome stability during undisturbed replication and in response to DNA damage. A ubiquitin-binding zinc finger (UBZ) domain directs human WRNIP1 to nuclear foci, but the functional significance of its presence and the relevant ubiquitylation targets that this domain recognizes have remained unknown. Here, we provide a mechanistic basis for the ubiquitin-binding properties of the protein. We show that in yeast an analogous domain exclusively mediates the damage-related activities of Mgs1. By means of preferential physical interactions with the ubiquitylated forms of the replicative sliding clamp, proliferating cell nuclear antigen (PCNA), the UBZ domain facilitates recruitment of Mgs1 to sites of replication stress. Mgs1 appears to interfere with the function of polymerase δ, consistent with our observation that Mgs1 inhibits the interaction between the polymerase and PCNA. Our identification of Mgs1 as a UBZ-dependent downstream effector of ubiquitylated PCNA suggests an explanation for the ambivalent role of the protein in damage processing.

Bacterial chromosome replication is initiated by binding of DnaA to a DnaA-box cluster (DBC) within the replication origin (oriC). In Bacillus subtilis, six additional DBCs are found outside of oriC and some are known to be involved in transcriptional regulation of neighboring genes. A deletion mutant lacking the six DBCs (Δ6) initiated replication early. Further, inactivation of spo0J in Δ6 cells yielded a pleiotropic phenotype, accompanied by severe growth inhibition. However, a spontaneous suppressor in soj or a deletion of soj, which stimulates DnaA activity in the absence of Spo0J, counteracted these effects. Such abnormal phenotypic features were not observed in a mutant background in which replication initiation was driven by a plasmid-derived replication origin. Moreover, introduction of a single DBC at various ectopic positions within the Δ6 chromosome partly suppressed the early-initiation phenotype, but this was dependent on insertion location. We propose that DBCs negatively regulate replication initiation by interacting with DnaA molecules and play a major role, together with Spo0J/Soj, in regulating the activity of DnaA.

This report investigates the mechanisms by which mammalian cells coordinate DNA replication with transcription and chromatin assembly. In yeast, DNA replication initiates within nucleosome-free regions, but studies in mammalian cells have not revealed a similar relationship. Here, we have used genome-wide massively parallel sequencing to map replication initiation events, thereby creating a database of all replication initiation sites within nonrepetitive DNA in two human cell lines. Mining this database revealed that genomic regions transcribed at moderate levels were generally associated with high replication initiation frequency. In genomic regions with high rates of transcription, very few replication initiation events were detected. High-resolution mapping of replication initiation sites showed that replication initiation events were absent from transcription start sites but were highly enriched in adjacent, downstream sequences. Methylation of CpG sequences strongly affected the location of replication initiation events, whereas histone modifications had minimal effects. These observations suggest that high levels of transcription interfere with formation of pre-replication protein complexes. Data presented here identify replication initiation sites throughout the genome, providing a foundation for further analyses of DNA–replication dynamics and cell-cycle progression.

Ribonucleoside diphosphate reductase (RNR) is located in discrete foci in a number that increases with the overlapping of replication cycles in Escherichia coli. Comparison of the numbers of RNR, DnaX and SeqA protein foci with the number of replication forks at different growth rates reveals that fork : focus ratios augment with increasing growth rates, suggesting a higher cohesion of the three protein foci with increasing number of forks per cell. Quantification of NrdB and SeqA proteins per cell showed: (i) a higher amount of RNR per focus at faster growth rates, which sustains the higher cohesion of RNR foci with higher numbers of forks per cell; and (ii) an equivalent amount of RNR per replication fork, independent of the number of the latter.

Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly,Escherichia colicells carrying thenrdA101allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 °C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 °C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 °C cannot explain the completion of chromosomal DNA replication in thenrdA101mutant. We found that inactivation of the DnaA protein by using severaldnaAtsalleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in thenrdA101mutant at 42 °C. As both inactivation of the DnaA protein and inhibition of RNA synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of forks per chromosome could be related to those effects. In support of this notion, we found that avoiding multifork replication rounds by the presence of moderate extra copies ofdatAsequence increases the relative amount of DNA synthesis of thenrdA101mutant at 42 °C. We propose that a lower replication fork density results in an improvement of the progression of DNA replication, allowing replication of the entire chromosome at the restrictive temperature. The mechanism related to this effect is also discussed.

In addition to their role in the virulence attenuation of Salmonella and other pathogens, dam or seqA genes increase the sensitivity towards hydrogen peroxide. The aim of our study is to investigate the effect of H(2)O(2) on the motility, the catalase and superoxide dismutase activities of dam and/or seqA mutants of Salmonella typhimurium. Our findings showed significant differences of the effects of H(2)O(2) on the motility between wild type strain and all of mutants. Hydrogen peroxide changes SOD isoenzyme profile of these mutants by disappearance of Fe-SOD. Concerning the catalase, an increase of its activity was observed in the wild type, dam and seqA mutant. However, H(2)O(2) decreases the activity of this enzyme in the double mutant strain. We can suggest that the dam gene, together with seqA, play a protective role in the oxidative stress response of Salmonella typhimurium.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.

We have investigated the replication patterns of the two chromosomes of the bacterium Vibrio cholerae grown in four different media. By combining flow cytometry and quantitative real-time PCR with computer simulations, we show that in rich media, V. cholerae cells grow with overlapping replication cycles of both the large chromosome (ChrI) and the small chromosome (ChrII). In Luria–Bertani (LB) medium, initiation occurs at four copies of the ChrI origin and two copies of the ChrII origin. Replication of ChrII was found to occur at the end of the ChrI replication period in all four growth conditions. Novel cell-sorting experiments with marker frequency analysis support these conclusions. Incubation with protein synthesis inhibitors indicated that the potential for initiation of replication of ChrII was present at the same time as that of ChrI, but was actively delayed until much of ChrI was replicated. Investigations of the localization of SeqA bound to new DNA at replication forks indicated that the forks were co-localized in pairs when cells grew without overlapping replication cycles and in higher-order structures during more rapid growth. The increased degree of fork organization during rapid growth may be a means by which correct segregation of daughter molecules is facilitated.

We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.

Most of the core components of the archaeal chromosomal DNA replication apparatus share significant protein sequence similarity with eukaryotic replication factors, making the Archaea an excellent model system for understanding the biology of chromosome replication in eukaryotes. The present review summarizes current knowledge of how the core components of the archaeal chromosome replication apparatus interact with one another to perform their essential functions.

Previously, inter-chromosomal interactions called “chromosome kissing” have been reported to control tissue-specific transcription and cell fate determination. Using the fission yeast as a model system we have shown that physiologically programmed replication termination is also modulated by chromosome kissing. The published report reviewed here shows that a myb-like replication terminator protein Reb1 of S. pombe and its cognate binding sites (Ter) are involved in chromosome kissing that promotes a cooperative mechanism of replication termination. We also suggest that at least one other replication terminator protein namely Sap1, which is also an origin binding protein, is likely to be involved in a similar mechanism of control not only of fork arrest but also of replication initiation and in possible ori-Ter interaction. We discuss the roles of chromatin remodeling and other proteins in this novel mechanism of replication control.

The pBR322 plasmid origin replication and oriC show similar responses to adenine methylation. Both are subject to sequestration by membrane fractions. In fact, like the host origin oriC, the RNA II promoter region of pBR322 is regulated by methylation of three GATC adenine methylation sites. The SeqA gene product acts in the negative control of oriC by sequestration. We suggest that the role of SeqA protein in sequestration is similar to oriC region DNA. Hence, SeqA recognize the methylation state of the pBR322RNA II promoter region by direct DNA binding in vitro . Using the pOC42 plasmid, we show that SeqA binds exclusively to the hemimethylated form of the replication origin of the pBR322 plasmid. In addition, we suggested that the SeqA protein could modulate periodically the initiation of replication of the pBR322 plasmid. The later could be fixed by its origin sequence, on a hemimethylated state, during the initiation of the replication.

Summary Chromosome replication in Escherichia coli is limited by the supply of DnaA associated with ATP. Cells deficient in RIDA (Regulatory Inactivation of DnaA) due to a deletion of the hda gene accumulate suppressor mutations (hsm) to counteract the overinitiation caused by an elevated DnaAATP level. Eight spontaneous hda suppressor mutations were identified by whole-genome sequencing, and three of these were analysed further. Two mutations (hsm-2 and hsm-4) mapped in the dnaA gene and led to a reduced ability to initiate replication from oriC. One mutation (hsm-1) mapped to the seqA promoter and increased the SeqA protein level in the cell. hsm-1 cells had prolonged origin sequestration, reduced DnaA protein level and reduced DnaA-Reactivating Sequence (DARS)-mediated rejuvenation of DnaAADP to DnaAATP, all of which could contribute to the suppression of RIDA deficiency. Despite of these defects hsm-1 cells were quite similar to wild type with respect to cell cycle parameters. We speculate that since SeqA binding sites might overlap with DnaA binding sites spread throughout the chromosome, excess SeqA could interfere with DnaA titration and thereby increase free DnaA level. Thus, in spite of reduction in total DnaA, the amount of DnaA molecules available for initiation may not be reduced.

The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage single-stranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single- and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.

We successfully substituted Escherichia coli 's origin of replication oriC with the origin region of Vibrio cholerae chromosome I ( oriCI Vc ). Replication from oriCI Vc initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC . With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI Vc were similar. Since Hda is involved in the conversion of DnaA ATP (DnaA bound to ATP) to DnaA ADP (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli . No hda homologue has been identified in V. cholerae yet. In V. cholerae , dam is essential for viability, whereas in E. coli , dam mutants are viable. Replacement of E. coli oriC with oriCI Vc allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI Vc . We show that when E. coli 's origin of replication is substituted by oriCI Vc , dam , but not seqA , becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI Vc in E. coli . In this model, methylation at oriCI Vc would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.

DNA replication in Escherichia coli is initiated by DnaA binding to oriC, the replication origin. During the process of assembly of the replication factory, the DnaA is released back into the cytoplasm, where it is competent to reinitiate replication. Premature reinitiation is prevented by binding SeqA to newly formed GATC sites near the replication origin. Resolution of the resulting SeqA cluster is one aspect of timing for reinitiation. A Markov model accounting for the competition between SeqA binding and methylation for one or several GATC sites relates the timing to reaction rates, and consequently to the concentrations of SeqA and methylase. A model is proposed for segregation, the motion of the two daughter DNAs into opposite poles of the cell before septation. This model assumes that the binding of SeqA and its subsequent clustering results in loops from both daughter nucleoids attached to the SeqA cluster at the GATC sites. As desequestration occurs, the cluster is divided in two, one associated with each daughter. As the loops of DNA uncoil, the two subclusters migrate apart due to the Brownian ratchet effect of the DNA loop.

Gene HSM3 encodes the Hsm3 protein involved in the minor branch in the system responsible for the correction of mismatched bases in DNA structure and controls replicative and reparative spontaneous mutagenesis in yeast Saccharomyces cerevisiae. Spontaneous and UV-induced mutagenesis was studied in three mutant alleles of gene HSM3, and repair effectivity of artificial heteroduplexes was assessed in DNA molecule. The resuts of these studies allowed establishment of the protein domain structure of protein Hsm3 and functions of each domain: the N-terminal domain is responsible for binding to mispaired bases, and the C-terminal domain ensures the interaction with other proteins involved in the system of mismatched base correction.

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP-ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism.

Phase variation of the outer membrane protein Ag43 encoded byagn43inEscherichia coliis controlled by an epigenetic mechanism. Sequestration of the regulatory region from Dam-dependent methylation has to be established and maintained throughout a generation to obtain and maintain the OFF phase. This work shows that hemimethylated DNA, which is formed by the passage of the DNA replication fork in an ON-phase cell, can be sequestered from methylation by OxyR binding, which is thus a key event for the switch from ON to OFF. No evidence was found that the protein SeqA, which also binds to the region, is involved in sequestration. To facilitate the dissection of this process further, a novel approach was introduced that does not alter the sequence of the regulatory region or the cellular concentration of Dam or OxyR, which consists of inserting auxiliary OxyR binding sites upstream of the regulatory region. Using this strategy, it was shown that the ON-to-OFF switch frequency can be modulated without changing the OFF-to-ON frequency. The data support a model in which in an ON-phase cell, the subcellular OxyR availability at the replication fork as it passes through theagn43regulatory region is key for initiating an ON-to-OFF switch. In contrast, this availability is not a determining factor for the switch from OFF to ON. This finding shows that different variables affect these two stochastic events. This provides new insight into the events determining the stochastic nature of epigenetic phase variation.

Expression of Epstein-Barr Virus BZLF1, a key protein initiating the switch from latent to lytic infection, is known to cause cell growth arrest by accumulating p53 and p21(WAF1/CIP1) in epithelial cells, but its molecular mechanism remains elusive. We found here that the BZLF1 protein stimulates p53 binding to its recognition sequence. The BZLF1 accelerated the rate of p53-DNA complex formation through the interaction with p53 protein and also enhanced p53-specific transcription in vitro. Furthermore, p53 protein was found to bind to its target promoter regions specifically in the early stages of lytic replication. Overexpression of p53 at the early stages of lytic replication enhanced viral genome replication, supporting the idea that p53 plays an important role in the initiation steps of EBV replication. Taking the independent role of BZLF1 on p53 degradation into consideration, we propose that the BZLF1 protein regulates p53 and its target gene products in two distinctive manners; transient induction of p53 at the early stages for the initiation of viral productive replication and p53 degradation at the later stages for S-phase like environment preferable for viral replication.

Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a unique nucleoid-associated protein that binds AT-rich regions of the genome, including genomic islands acquired by horizontal gene transfer and regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM and PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain-swapping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA-binding domain. Nuclear magnetic resonance analysis of the DNA-binding domain of Lsr2 and its interaction with DNA reveals a unique structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve a function similar to the Enterobacteriaceae H-NS, likely coordinating global gene regulation and virulence in this group of medically important bacteria.

January 1, 2011, The essential proteins DnaB, DnaD and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order. We used temperature-sensitive mutants or a conditional degradation system to inactivate each protein and test for association of the other proteins with oriC in vivo. We found that there was a clear order of stable association with oriC; DnaA, DnaD, DnaB, and finally DnaI-mediated loading of helicase. The loading of helicase via stable intermediates resembles that of eukaryotes and the established hierarchy provides several potential regulatory points. The general approach described here can be used to analyse assembly of other complexes., Netherlands Organization for Scientific Research (Rubicon fellowship), National Institutes of Health (U.S.) (Public Health Service Grant GM41934)

Replication initiation is a key event in the cell cycle of all organisms and oriC, the replication origin in Escherichia coli, serves as the prototypical model for this process. The minimal sequence required for oriC function was originally determined entirely from plasmid studies using cloned origin fragments, which have previously been shown to differ dramatically in sequence requirement from the chromosome. Using an in vivo recombineering strategy to exchange wt oriCs for mutated ones regardless of whether they are functional origins or not, we have determined the minimal origin sequence that will support chromosome replication. Nearly the entire right half of oriC could be deleted without loss of origin function, demanding a reassessment of existing models for initiation. Cells carrying the new DnaA box-depleted 163 bp minimal oriC exhibited little or no loss of fitness under slow-growth conditions, but were sensitive to rich medium, suggesting that the dense packing of initiator binding sites that is a hallmark of prokaryotic origins, has likely evolved to support the increased demands of multi-forked replication.

The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.

The replication protein ORF904 from the plasmid pRN1 is a multifunctional enzyme with ATPase-, primase- and DNA polymerase activity. Sequence analysis suggests the presence of at least two conserved domains: an N-terminal prim/pol domain with primase and DNA polymerase activities and a C-terminal superfamily 3 helicase domain with a strong double-stranded DNA dependant ATPase activity. The exact molecular function of the helicase domain in the process of plasmid replication remains unclear. Potentially this motor protein is involved in duplex remodelling and/or origin opening at the plasmid replication origin. In support of this we found that the monomeric replication protein ORF904 forms a hexameric ring in the presence of DNA. It is able to translocate along single-stranded DNA in 3'-5' direction as well as on double-stranded DNA. Critical residues important for ATPase activity and DNA translocation activity were identified and are in agreement with a homology model of the helicase domain. In addition we propose that a winged helix DNA-binding domain at the C-terminus of the helicase domain could assist the binding of the replication protein specifically to the replication origin.

The initiation of replication in bacteria is regulated via the initiator protein DnaA. ATP-bound DnaA binds to multiple sequences at the origin of replication, oriC, unwinding the DNA and promoting the binding of DnaB helicase. From an Escherichia coli mutant highly perturbed for replication control, obgE∷Tn5-EZ seqAΔ, we isolated multiple spontaneous suppressor mutants with enhanced growth and viability. These suppressors suppressed the replication control defects of mutants in seqA alone and genetically mapped to the essential dnaA replication initiator gene. DNA sequence analysis of four independent isolates revealed an identical deletion of the DnaA-coding region at a repeated hexanucleotide sequence, causing a loss of 25 amino acids in domain II of the DnaA protein. Previous work has established no function for this region of protein, and deletions in the region, unlike other domains of the DnaA protein, do not produce lethality. Flow cytometric analysis established that this allele, dnaAΔ96-120, ameliorated the over-replication phenotype of seqA mutants and reduced the DNA content of wild-type strains; virtually identical effects were produced by loss of the DnaA-positive regulatory protein DiaA. DiaA binds to multiple DnaA subunits and is thought to promote cooperative DnaA binding to weak affinity DNA sites through interactions with DnaA in domains I and/or II. The dnaAΔ96-120 mutation did not affect DiaA binding in pull-down assays, and we propose that domain II, like DiaA, is required to promote optimal DnaB recruitment to oriC.

Epstein-Barr virus nuclear antigen 1 (EBNA1) is a viral protein required for stable replication and segregation of DNA episomes containing the Epstein-Barr virus (EBV) origin of replication, OriP. Overproduction of EBNA1 protein in Escherichia coli has previously been shown to be difficult due to the large number of codons in EBNA1 gene that are infrequently used in E. coli. Here we changed the 26 rare codons that are found among the first 78 codons of EBNA1 gene, and replaced them with codons that encode the same amino-acids but are abundant in E. coli. This led to a significant improvement of EBNA1 expression in a standard E. coli strain. Partial EBNA1 polypeptides of 11.5-16 kDa extending from the N-terminus to the second arginine and glycine-rich region were extremely abundant in the extract, however, resulting in a second limitation of the level of EBNA1 expression. EBNA1 was expressed as a fusion with a C-terminal six-histidine tag in order to get rid of the short polypeptides by Ni-NTA affinity purification, and salt conditions were used that allowed us to extract and purify EBNA1 without resorting to protein denaturing reagents. The purified protein was used in DNA-binding experiments with DNA fragments containing specific EBNA1 sites. The E. coli-expressed protein formed specific DNA-protein complexes that could be analyzed in polyacrylamide gels without showing the aggregation, or linking, phenomenon that is usually observed with EBNA1 expressed in eukaryotic cells. EBNA1 protein expressed in E. coli should therefore prove useful to further study the biochemical properties of this crucial Epstein-Barr virus protein.

The efficacy of a vaccine is conditioned by its capacity to elicit a protective immune response. The principal safety concerns of live vaccine are virulence reversion. The aim of this work was to evaluate the virulence of Salmonella typhimurium seqA mutant after cohabitation with mice. Our results indicated that LD50 of hosted strains were at least twofold lower than those of parental strains. Also, the in vivo competition assays have showed that the development of a systemic infection was most obvious for recovered strains than for control strains. In addition, the number of hosted mutants colonizing spleen and liver was relatively higher than control strains. Adhesion and invasion experiments were performed in order to compare the pathogenicity of Salmonella. For instance recovered-mutant attached to epithelial cells (KB cells) better than parental strains. According to these results, we report that in vivo adaptation of Salmonella typhimurium seqA mutants can increase their virulence.

EhMLBP is an essential Entamoeba histolytica protein that binds preferentially to methylated long interspersed nuclear elements and rDNA. In an effort to identify more EhMLBP DNA substrates, we developed an affinity-based technique in which the C-terminal DNA binding domain of EhMLBP (GST-CterEhMLBP) was used as the ligand. Bioinformatic analysis of the DNA sequences that were isolated by this affinity method revealed the presence of a 29-nucleotide consensus motif that includes a stretch of ten adenines. Gel retardation analysis showed that EhMLBP binds to the consensus motif with a preference for its methylated form. Four DNA sequences, namely those that encoded either dihydrouridine synthetase, RAP GTPase activating protein, serine/threonine protein kinase or leucine-rich repeat containing protein (LRPP) were then selected for further analysis. In vivo binding of EhMLBP to these genes was confirmed by chromatin immunoprecipitation. The presence of methylated cytosines was detected in DNA encoding LRPP and to a lower extent in the other genes. EhMLBP binds preferentially to the methylated forms of these DNA targets. The ability of the consensus motif to compete with EhMLBP binding to its DNA substrates indicates that the adenine stretch is involved in the mechanism of DNA recognition. The results of this investigation extend our existing knowledge on the number of DNA sequences that are recognized by EhMLBP and reinforce the notion that this protein is an innate methylated DNA binding protein in E. histolytica.

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in livingEscherichia colicells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of theoriCregions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from theoriCregion. However, involvement ofdatAin the initiation control was suggested from the observation that, inΔdatAcells, DnaA-EYFP maximally colocalized with theoriCregion earlier in the cell cycle than it did in wild-type cells andoriCconcentration was increased.

INI1/hSNF5/BAF47/SMARCB1 is an HIV-1 integrase (IN)-binding protein that modulates viral replication in multiple ways. A minimal IN-binding domain of INI1, S6 (amino acids 183–294), transdominantly inhibits late events, and down-modulation of INI1 stimulates early events of HIV-1 replication. INI1 both stimulates and inhibits in vitro integration depending on IN concentration. To gain further insight into its role in HIV-1 replication, we purified and biochemically characterized INI1. We found that INI1 forms multimeric structures. Deletion analysis indicated that the Rpt1 and Rpt2 motifs form the minimal multimerization domain. We isolated mutants of INI1 that are defective for multimerization using a reverse yeast two-hybrid system. Our results revealed that INI1 residues involved in multimerization overlap with IN-binding and nuclear export domains and are required for nuclear retention and co-localization with IN. Multimerization-defective mutants are also defective for mediating the transdominant effect of INI1-S6-(183–294). Furthermore, we found that INI1 is a minor groove DNA-binding protein. Although IN binding and multimerization are required for INI1-mediated inhibition, the acceptor DNA binding property of INI1 may be required for stimulation of in vitro strand transfer activities of IN. Binding of INI1 to IN results in the formation of presumably inactive high molecular weight IN-INI1 complexes, and the multimerization-defective mutant was unable to form these complexes. These results indicate that the multimerization and IN binding properties of INI1 are necessary for its ability to both inhibit integration and influence assembly and particle production, providing insights into the mechanism of INI1-mediated effects in HIV-1 replication.

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential in DNA metabolism and is phosphorylated in response to DNA-damaging agents. Rad52 and RPA participate in the repair of double-stranded DNA breaks (DSBs). It is known that human RPA and Rad52 form a complex, but the molecular mass, stoichiometry, and exact role of this complex in DSB repair are unclear. In this study, absolute molecular masses of individual proteins and complexes were measured in solution using analytical size-exclusion chromatography coupled with multiangle light scattering, the protein species present in each purified fraction were verified via sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE)/Western analyses, and the presence of biotinylated ssDNA in the complexes was verified by chemiluminescence detection. Then, employing UV cross-linking, the protein partner holding the ssDNA was identified. These data show that phosphorylated RPA promoted formation of a complex with monomeric Rad52 and caused the transfer of ssDNA from RPA to Rad52. This suggests that RPA phosphorylation may regulate the first steps of DSB repair and is necessary for the mediator function of Rad52.

The seqA defect in Escherichia coli increases the ori/ter ratio and causes chromosomal fragmentation, making seqA mutants dependent on recombinational repair (the seqA recA colethality). To understand the nature of this chromosomal fragmentation, we characterized DeltaseqA mutants and isolated suppressors of the DeltaseqA recA lethality. We demonstrate that our DeltaseqA alleles have normal function of the downstream pgm gene and normal ratios of the major phospholipids in the membranes, but increased surface lipopolysaccharide (LPS) phosphorylation. The predominant class of DeltaseqA recA suppressors disrupts the rfaQGP genes, reducing phosphorylation of the inner core region of LPS. The rfaQGP suppressors also reduce the elevated ori/ter ratio of the DeltaseqA mutants but, unexpectedly, the suppressed mutants still exhibit the high levels of chromosomal fragmentation and SOS induction, characteristic of the DeltaseqA mutants. We also found that colethality of rfaP with defects in the production of acidic phospholipids is suppressed by alternative initiation of chromosomal replication, suggesting that LPS phosphorylation stimulates replication initiation. The rfaQGP suppression of the seqA recA lethality provides genetic support for the surprising physical evidence that the oriC DNA forms complexes with the outer membrane.

Summary Mutants of dnaAcos are inviable at 30°C because DnaAcos hyperinitiates, leading to new replication forks that apparently collide from behind with stalled forks, thereby generating lethal double-strand breaks. By comparison, an elevated level of DnaA also induces extra initiations, but lethality occurs only in strains defective in repairing double-strand breaks. To explore the model that the chromosomal level of DnaAcos, or the increased abundance of DnaA, increases initiation frequency by, escaping or overcoming pathways that control initiation, respectively, we developed a genetic selection and identified seqA, datA, dnaN and hda, which function in pathways that either act at oriC or modulate DnaA activity. To assess each pathway's relative effectiveness, we used genetically inactivated strains, and quantified initiation frequency after elevating the level of DnaA. The results indicate that the hda-dependent pathway has a stronger effect on initiation than pathways involving seqA and datA. Testing the model that DnaAcos overinitiates because it fails to respond to one or more regulatory mechanisms, we show that dnaAcos is unresponsive to hda and dnaN, which encodes the β clamp, and also datA, a locus proposed to titer excess DnaA. These results explain how DnaAcos hyperinitiates to interfere with viability.

When the bacterium Escherichia coli is grown in rich medium, the replication and segregation periods may span two, three or four generations and cells may contain up to 24 replication forks. The newly synthesized, hemimethylated DNA at each fork is bound by SeqA protein. The SeqA-DNA structures form distinct foci that can be observed by immunofluorescence microscopy. The numbers of foci were lower than the numbers of replication forks indicating fork co-localization. The extent of co-localization correlated with the extent of replication cycle overlap in wild-type cells. No abrupt increase in the numbers of foci occurred at the time of initiation of replication, suggesting that new replication forks bind to existing SeqA structures. Manipulations with replication control mechanisms that led to extension or reduction of the replication period and number of forks, did not lead to changes in the numbers of SeqA foci per cell. The results indicate that the number of SeqA foci is not directly governed by the number of replication forks, and supports the idea that new DNA may be 'captured' by existing SeqA structures.

The Escherichia coli SeqA protein contributes to regulation of chromosome replication by preventing re-initiation at newly replicated origins. SeqA protein binds to new DNA which is hemimethylated at the adenine of GATC sequences. Most of the cellular SeqA is found complexed with the new DNA at the replication forks. In vitro the SeqA protein binds as a dimer to two GATC sites and is capable of forming a helical fiber of dimers through interactions of the N-terminal domain. SeqA can also bind, with less affinity, to fully methylated origins and affect timing of "primary" initiations. In addition to its roles in replication, the SeqA protein may also act in chromosome organization and gene regulation.

The ability to perform accurate sequential movements is essential to normal motor function. Learning a sequential motor behavior is comprised of two basic components: explicit identification of the order in which the sequence elements should be performed and implicit acquisition of spatial accuracy for each element. Here we investigated the time course of learning of these components for a first sequence (SEQA) and their susceptibility to interference from learning a second sequence (SEQB). We assessed explicit learning with a discrete index, the number of correct anticipatory movements, and implicit learning with a continuous variable, spatial error, which decreased during learning without subject awareness. Spatial accuracy to individual sequence elements reached asymptotic levels only when the whole sequence order was known. Interference with recall of the order of SEQA persisted even when SEQB was learned 24 h after SEQA. However, there was resistance to interference by SEQB with increased initial training with SEQA. For implicit learning of spatial accuracy, SEQB interfered at 5 min but not 24 h after SEQA. As in the case of sequence order, prolonged initial training with SEQA induced resistance to interference by SEQB. We conclude that explicit sequence learning is more susceptible to anterograde interference and implicit sequence learning is more susceptible to retrograde interference. However, both become resistant to interference with saturation training. We propose that an essential feature of motor skill learning is the process by which discrete explicit task elements are combined with continuous implicit features of movement to form flawless sequential actions.

In Escherichia coli, ATP-DnaA, unlike ADP-DnaA, can initiate chromosomal replication at oriC. The level of cellular ATP-DnaA fluctuates, peaking at around the time of replication initiation. However, it remains unknown how the ATP-DnaA level increases coordinately with the replication cycle. In this study, we show that two chromosomal intergenic regions, herein termed DnaA-reactivating sequence 1 (DARS1) and DnaA-reactivating sequence 2 (DARS2), directly promote regeneration of ATP-DnaA from ADP-DnaA by nucleotide exchange, resulting in the promotion of replication initiation in vitro and in vivo. Coordination of initiation with the cell cycle requires DARS activity and its regulation. Oversupply of DARSs results in increase in the ATP-DnaA level and enhancement of replication initiation, which can inhibit cell growth in an oriC-dependent manner. Deletion of DARSs results in decrease in the ATP-DnaA level and inhibition of replication initiation, which can cause synthetic lethality with a temperature-sensitive mutant dnaA and suppression of overinitiation by the lack of seqA or datA, negative regulators for initiation. DARSs bear a cluster of DnaA-binding sites. DnaA molecules form specific homomultimers on DARS1, which causes specific interactions among the protomers, reducing their affinity for ADP. Our findings reveal a novel regulatory pathway that promotes the initiation of chromosomal replication via DnaA reactivation.

SeqA is a negative regulator of DNA replication in Escherichia coli and related bacteria that functions by sequestering the origin of replication and facilitating its resetting after every initiation event. Inactivation of the seqA gene leads to unsynchronized rounds of replication, abnormal localization of nucleoids and increased negative superhelicity. Excess SeqA also disrupts replication synchrony and affects cell division. SeqA exerts its functions by binding clusters of transiently hemimethylated GATC sequences generated during replication. However, the molecular mechanisms that trigger formation and disassembly of such complex are unclear. We present here the crystal structure of a dimeric mutant of SeqA [SeqADelta(41-59)-A25R] bound to tandem hemimethylated GATC sites. The structure delineates how SeqA forms a high-affinity complex with DNA and it suggests why SeqA only recognizes GATC sites at certain spacings. The SeqA-DNA complex also unveils additional protein-protein interaction surfaces that mediate the formation of higher ordered complexes upon binding to newly replicated DNA. Based on this data, we propose a model describing how SeqA interacts with newly replicated DNA within the origin of replication and at the replication forks.

The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer alpha-beta sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein-Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.

Relatively little is known about the interaction of eukaryotic replication terminator proteins with the cognate termini and the replication termination mechanism. Here, we report a biochemical analysis of the interaction of the Reb1 terminator protein of Schizosaccharomyces pombe, which binds to the Ter3 site present in the nontranscribed spacers of ribosomal DNA, located in chromosome III. We show that Reb1 is a dimeric protein and that the N-terminal dimerization domain of the protein is dispensable for replication termination. Unlike its mammalian counterpart Ttf1, Reb1 did not need an accessory protein to bind to Ter3. The two myb/SANT domains and an adjacent, N-terminal 154-amino-acid-long segment (called the myb-associated domain) were both necessary and sufficient for optimal DNA binding in vitro and fork arrest in vivo. The protein and its binding site Ter3 were unable to arrest forks initiated in vivo from ars of Saccharomyces cerevisiae in the cell milieu of the latter despite the facts that the protein retained the proper affinity of binding, was located in vivo at the Ter site, and apparently was not displaced by the “sweepase” Rrm3. These observations suggest that replication fork arrest is not an intrinsic property of the Reb1-Ter3 complex.

In order to explore the mechanism of geminivirus DNA replication, we show that the Replication initiator (Rep) protein encoded by Mungbean yellow mosaic India virus (MYMIV), a member of the family Geminiviridae, binds specifically to the iterons present in the viral DNA replication origin (CR-A) in a highly ordered manner that might be a prerequisite for the initiation of replication. MYMIV Rep also acts as a helicase during the post-initiation stage and is upregulated in presence of the RPA32 subunit of Replication Protein A. The implication of these findings on the initiation and elongation stages of MYMIV DNA replication has been discussed.

Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli . While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli , newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae , suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.

The E2 proteins of several papillomaviruses link the viral genome to mitotic chromosomes to ensure retention and the efficient partitioning of genomes into daughter cells following cell division. Bovine papillomavirus type 1 E2 binds to chromosomes in a complex with Brd4, a cellular bromodomain protein. Interaction with Brd4 is also important for E2-mediated transcriptional regulation. The transactivation domain of E2 is crucial for interaction with the Brd4 protein; proteins lacking or mutated in this domain do not interact with Brd4. However, we found that the C-terminal DNA binding/dimerization domain of E2 is also required for efficient binding to Brd4. Mutations that eliminated the DNA binding function of E2 had no effect on the ability of E2 to interact with Brd4, but an E2 protein with a mutation that disrupted C-terminal dimerization bound Brd4 with greatly reduced efficiency. Furthermore, E2 proteins in which the C-terminal domains were replaced with the dimeric DNA binding domain of EBNA-1 or Gal4 bound efficiently to the Brd4 protein, but the replacement of the E2 C-terminal domain with a monomeric red fluorescent protein did not rescue efficient Brd4 binding. Thus, E2 bound to Brd4 most efficiently as a dimer. To prove this finding further, the E2 DNA binding domain was replaced with an FKBP12-derived domain in which dimerization was regulated by a bivalent ligand. This fusion protein bound Brd4 efficiently only in the presence of the ligand, confirming that a dimer of E2 was required. Correspondingly, E2 proteins that could dimerize were able to bind to mitotic chromosomes much more efficiently than monomeric E2 polypeptides.

Faithful replication of the entire genome once per cell cycle is essential for maintaining genetic integrity, and the origin of DNA replication is key in this regulation. Unlike that in unicellular organisms, the replication initiation mechanism in mammalian cells is not well understood. We have identified a strong origin of replication at the DBF4 promoter locus, which contains two initiation zones, two origin recognition complex (ORC) binding sites and two DNase I-hypersensitive regions within approximately 1.5 kb. Notably, similar to the Escherichia coli oriC, replication at the DBF4 locus starts from initiation zone I, which contains an ORC-binding site, and progresses in the direction of transcription toward initiation zone II, located approximately 0.4 kb downstream. Replication on the opposite strand from zone II, which contains another ORC-binding site, may be activated or facilitated by replication from zone I. We term this new mammalian replication mode 'asymmetric bidirectional replication'.

Papillomavirus genomes replicate as nuclear plasmids at a low copy number in undifferentiated keratinocytes. Papillomaviruses encode the E1 and E2 proteins that bind to the origin of replication and are required for the activation of replication. In addition to E2, several papillomaviruses express an E8⁁E2C protein, which is generated by alternative splicing and functions as a transcriptional repressor and inhibitor of the E1/E2-dependent replication of the viral origin. Previous analyses suggested that the E8 domain functions as a transferable repression domain. In this report we present evidence that the E8 domain is responsible for the interaction with cellular corepressor molecules such as histone deacetylases, the histone methyltransferase SETDB1, and the TRIM28/KAP-1/TIF1β/KRIP-1 protein. Whereas the interaction with histone deacetylases is involved only in transcriptional repression, the interaction with TRIM28/KAP-1/TIF1β/KRIP-1 contributes to the inhibition of E1/E2-dependent replication. The corepressor TRIM28/KAP-1/TIF1β/KRIP-1 has been described to be part of multicomponent complexes involved in transcriptional regulation and functions as a scaffold protein. Since neither histone deacetylases nor the histone methyltransferase SETDB1 appears to be involved in the inhibition of E1/E2-dependent replication, most likely the modification of non-histone proteins contributes to the replication repression activity of E8⁁E2C.