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PubMed1997

The efficiency and timing of initiation of replication of multiple replicons of Saccharomyces cerevisiae chromosome VI.

BACKGROUND: A complete set of nine ARSs was identified (the tenth ARS in this paper), mapped on chromosome VI of Saccharomyces cerevisiae, and characterized for functional elements. RESULTS: The level of activity of all ARSs as chromosomal replication origins was determined by neutral/neutral 2D gel-electrophoresis. These origins were classified into three groups: (i) three high frequency origins used once nearly every cell cycle, (ii) four intermediate frequency origins used once in two to three cycles and (iii) two low frequency origins used in fewer than 5% of cell cycles. These variations in initiation frequency among origins of chromosome VI are present in three common laboratory wild-type strains. Each origin is initiated at a fixed time and passively replicated by incoming replication forks at a fixed time during a synchronous S phase. Replication of each arm of the chromosome starts from one major origin located one-fifth (left arm) and one-third (right arm) of the distance from the centromere, and expands sequentially in both directions. Two telomere vicinity origins are replicated last. Time of initiation and replication of the last replicating origin, Ori609, was remarkably variable from cell to cell. CONCLUSIONS: Chromosome VI of S. cerevisiae contains nine replication origins that comprise five active replicons under normal cell growth conditions. A clear correlation was found between the efficiency of initiation and the order of replication. The timing of initiation of most origins, except for the first and last, is coincident with the time of passive replication by incoming forks from neighbouring origins.

Cell Cycle↗

PubMed2005

Temporal profile of replication of human chromosomes.

Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that fire reproducibly. Early replication was correlated with high gene density. An acute transition of gene density from early to late replicating areas suggests that discrete chromatin states dictate early versus late replication. Surprisingly, at least 60% of the interrogated chromosomal segments replicate equally in all quarters of S phase, suggesting that large stretches of chromosomes are replicated by inefficient, variably located and asynchronous origins and forks, producing a pan-S phase pattern of replication. Thus, at least for aneuploid cancer cells, a typical discrete time of replication in S phase is not seen for large segments of the chromosomes.

Chromosomes, Human↗

PubMed2001

Bacteriophage T4 gene 41 helicase and gene 59 helicase-loading protein: a versatile couple with roles in replication and recombination.

Bacteriophage T4 uses two modes of replication initiation: origin-dependent replication early in infection and recombination-dependent replication at later times. The same relatively simple complex of T4 replication proteins is responsible for both modes of DNA synthesis. Thus the mechanism for loading the T4 41 helicase must be versatile enough to allow it to be loaded on R loops created by transcription at several origins, on D loops created by recombination, and on stalled replication forks. T4 59 helicase-loading protein is a small, basic, almost completely alpha-helical protein whose N-terminal domain has structural similarity to high mobility group family proteins. In this paper we review recent evidence that 59 protein recognizes specific structures rather than specific sequences. It binds and loads the helicase on replication forks and on three- and four-stranded (Holliday junction) recombination structures, without sequence specificity. We summarize our experiments showing that purified T4 enzymes catalyze complete unidirectional replication of a plasmid containing the T4 ori(uvsY) origin, with a preformed R loop at the position of the R loop identified at this origin in vivo. This replication depends on the 41 helicase and is strongly stimulated by 59 protein. Moreover, the helicase-loading protein helps to coordinate leading and lagging strand synthesis by blocking replication on the ori(uvsY) R loop plasmid until the helicase is loaded. The T4 enzymes also can replicate plasmids with R loops that do not have a T4 origin sequence, but only if the R loops are within an easily unwound DNA sequence.

DNA Helicases↗

PubMed2002

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.

Animals↗

PubMed2002

Replicational organization of three weakly expressed loci in Physarum polycephalum.

We previously mapped early-activated replication origins in the promoter regions of five abundantly transcribed genes in the slime mold Physarum polycephalum. This physical linkage between origins and genes is congruent with the preferential early replication of the active genes in mammalian cells. To determine how general this replicational organization is in the synchronous plasmodium of Physarum, we analyzed the replication of three weakly expressed genes. Bromodeoxyuridine (BrdUrd) density-shift and gene dosage experiments indicated that the redB (regulated in development) and redE genes replicate early, whereas redA replicates in mid-S phase. Bi-dimensional gel electrophoresis revealed that redA coincides with an origin that appears to be activated within a large temporal window in S phase so that the replication of the gene is not well defined temporally. The early replication of the redB and redE genes is due to the simultaneous activation of flanking origins at the onset of S phase. As a result, these two genes correspond to termination sites of DNA replication. Our data demonstrate that not all the Physarum promoters are preferred sites of initiation but, so far, all the expressed genes analyzed in detail either coincide with a replication origin or are embedded into a cluster of early firing replicons.

Animals↗

PubMed2005

Initiation of DNA replication at the human beta-globin 3' enhancer.

The origin of DNA replication in the human beta-globin gene contains an initiation region (IR) and two flanking auxiliary elements. Two replicator modules are located within the upstream auxiliary sequence and the IR core, but the functional sequences in the downstream auxiliary element are unknown. Here, we use a combination of benzoylated-naphthoylated DEAE (BND) cellulose purification and nascent strand abundance assays to show that replication initiation occurs at the beta-globin 3' enhancer on human chromosome 11 in the Hu11 hybrid murine erythroleukemia (MEL) cell line. To examine replicator function, 3' enhancer fragments were inserted into an ectopic site in MEL cells via an optimized FRT/EGFP-FLP integration system. These experiments demonstrate that the 1.6 kb downstream auxiliary element is a third replicator module called bGRep-E in erythroid cells. The minimal 260 bp 3' enhancer is required but not sufficient to initiate efficient replication, suggesting cooperation with adjacent sequences. The minimal 3' enhancer also cooperates with elements in an expressing HS3beta/gamma-globin construct to initiate replication. These data indicate that the beta-globin replicator has multiple initiation sites in three closely spaced replicator modules. We conclude that a mammalian enhancer can cooperate with adjacent sequences to create an efficient replicator module.

3' Flanking Region↗

PubMed2004

Dynamics of pre-replication complex proteins during the cell division cycle.

Replication of the human genome every time a cell divides is a highly coordinated process that ensures accurate and efficient inheritance of the genetic information. The molecular mechanism that guarantees that many origins of replication fire only once per cell-cycle has been the area of intense research. The origin recognition complex (ORC) marks the position of replication origins in the genome and serves as the landing pad for the assembly of a multiprotein, pre-replicative complex (pre-RC) at the origins, consisting of ORC, cell division cycle 6 (Cdc6), Cdc10-dependent transcript (Cdt1) and mini-chromosome maintenance (MCM) proteins. The MCM proteins serve as key participants in the mechanism that limits eukaryotic DNA replication to once-per-cell-cycle and its binding to the chromatin marks the final step of pre-RC formation, a process referred to as 'replication licensing'. We present data demonstrating how the MCM proteins associate with the chromatin during the G1 phase, probably defining pre-RCs and then anticipate replication fork movement in a precisely coordinated manner during the S phase of the cell cycle. The process of DNA replication must also be carefully coordinated with other cell-cycle processes including mitosis and cytokinesis. Some of the proteins that control initiation of DNA replication are likely to interact with the pathways that control these important cell-cycle transitions. Herein, we discuss the participation of human ORC proteins in other vital functions, in addition to their bona fide roles in replication.

Cell Cycle↗

PubMed1987

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells.

Simian virus 40 (SV40) DNA replication was studied in monolayers of infected monkey CV-1 cells, permeabilized with lysolecithin, by incubation with [alpha-32P]dTTP, the other dNTPs and rNTPs and an ATP-regenerating system. Analysis of the labeled SV40 DNA by sedimentation in alkaline sucrose gradients showed that about 30% of the material synthesized by the permeable cells in the course of 60 min consisted of covalently closed circular SV40 DNA (form I), with the remainder sedimenting as relaxed circles (form II) and replicative intermediates between 18 S and 4 S. The synthesis of SV40 DNA in the permeabilized cell system required the presence of all four dNTPs and was completely inhibited by aphidicolin, consistent with the involvement of DNA polymerase alpha. A detailed analysis of the distribution of radioactivity in the DNA synthesized involved cleavage with BstNI restriction endonuclease, followed by polyacrylamide gel electrophoresis and radioautography. The extent of labeling of all restriction fragments was nearly proportional to their length, suggesting that the entire SV40 chromosome was being replicated. This was confirmed by the careful comparison of the rate of labeling of a DNA fragment which includes the replication origin, and a fragment which includes the replication terminus. Their labeling was proportional to their size, regardless of the time for which the labeling was carried out. This demonstrated that the replication of the entire SV40 chromosome occurred in a steady state and that the start and termination of replication continuously occurred throughout the labeling period. The availability of an in vitro system in which replication of SV40 DNA undergoes multiple replication cycles should be of considerable value in the analysis of the mechanism of replication of this viral genome.

Animals↗

PubMed1981

Adenovirus DNA replication in vitro: a protein linked to the 5' end of nascent DNA strands.

Soluble nuclear extracts prepared from adenovirus-infected HeLa cells supported adenovirus DNA replication with exogenous DNA-protein complex as template, but protease-treated, phenol-extracted DNA was less active. Replication was enhanced when creatine phosphate and creatine phosphokinase were included in the reaction mixture, rendering the reaction independent of exogenous ATP. Genomic-length, newly synthesized DNA strands were first observed 30 min after initiation of replication and continued to increase in amount for at least 4 h. Thus, the rate of replication is consistent with previous estimates of the rate of replication in vivo. Nascent DNA strands bound to benzoylated, naphthoylated DEAE-cellulose due to their association with protein. The 5' termini of nascent DNA strands were resistant to the 5'- to 3'-specific T7 exonuclease, and the 3' termini of nascent strands were sensitive to the 3'- to 5'-specific exonuclease III. These results suggest that a protein becomes covalently linked to the 5' termini of nascent DNA strands replicated in vitro. Nuclear extracts prepared from adenovirus type 2-infected cells also supported replication of DNA-protein complex prepared from the unrelated type 7 adenovirus. The limited sequence homology between these two viruses at the origin of replication further defines recognition sequences at the origin. These results are discussed in terms of a model for adenovirus DNA replication in which the terminal protein and sequences within the inverted terminal repetition are involved in the formation of an initiation complex that is able to prime DNA replication.

Adenoviruses, Human↗

PubMed1982

Conserved sequences at the origin of adenovirus DNA replication.

The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A . T-rich region which is partially conserved among these serotypes, and a distal G . C-rich region which is less well conserved. The G . C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis.

Adenoviruses, Human↗

PubMed1987

Effects of position and orientation of the 72-base-pair-repeat transcriptional enhancer on replication from the simian virus 40 core origin.

A number of recent studies have reported that in papovaviruses such as simian virus 40 (SV40) and polyomavirus, the replication of the viral DNA in vivo is activated by the viral transcriptional enhancer or promoter sequences. Both viral and cellular transcriptional enhancers are well known for their ability to activate transcription in a position- and orientation-independent manner. In the present study, we investigated the effect of the position and orientation of the SV40 72-base-pair (bp) repeat enhancer on its replication activation function. We constructed plasmids containing one copy each of the SV40 core origin and enhancer placed in either order and orientation and at different distances from each other. We assayed the replication efficiencies of these plasmids in the presence of an internal control plasmid in COS-1 monkey kidney cells producing the SV40 T antigen required for replication. We found that the 72-bp repeat was capable of activating replication equally well in either orientation when placed 8 or 9 bp from the core origin. The activation of replication was totally abolished, and replication efficiencies in most instances were found to be lower than that obtained with the core origin alone, when the 72-bp repeat was separated from the core origin by distances of 99 bp or more. This was in direct contrast to the situation with polyomavirus, in which activation of replication by the homologous enhancer or by the SV40 72-bp repeat enhancer is known to be position independent. We also found that when the SV40 core origin and the 72-bp repeat enhancer were adjacent to each other, efficient activation of replication was obtained only if the end of the core origin containing the 17-bp A + T block was linked with the enhancer. In the other orientation of the core origin, activation of replication was either diminished or abolished. Hypotheses such as alteration of chromatin structure by the enhancer and interaction between trans-acting factors binding to the enhancer and the core origin mediating the activation effect are discussed.

Animals↗

PubMed1989

An element of the BK virus enhancer required for DNA replication.

The human papovavirus BK virus contains three 68-base-pair (bp) repeats that act as transcriptional enhancers. An analysis of plasmids containing the BK virus origin revealed that sequences within the 68-bp enhancer are required for DNA replication as well as transcription of the early promoter in COS-1 cells. Origins with a single 68-bp repeat replicated as efficiently as did those with three repeats when transfected into COS-1 cells. Replication did not occur in the absence of enhancer sequences and could not be restored by distal placement of enhancers to enhancerless origins. However, as with simian virus 40, replication in vitro was not dependent on the presence of any enhancer sequences. Deletion analysis showed that replication of BK virus origins was dependent on the presence of the first 21 bp of the enhancer contiguous with the A-T-rich stretch of the origin. This 21-bp element is referred to as the rep element. Although in combination with rep the remaining 47 bp of the enhancer appear to increase replication by two- to fivefold, they alone are not sufficient to support replication. Deletions or insertions in the enhancer which did not alter the rep element had no major effect on replication. Site-directed mutagenesis of the Sp1-like site within the rep element, the NF1 site present in the enhancer, or the NF1 site in adjacent late-side sequences each reduced transcription by two- to fivefold, but had no effect on replication, suggesting that replication and transcription can be uncoupled.

BK Virus↗

PubMed1994

Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.

ATP-Binding Cassette Transporters↗

PubMed1995

Two E2 binding sites alone are sufficient to function as the minimal origin of replication of human papillomavirus type 18 DNA.

Replication of papillomaviruses requires an origin of replication and two virus-encoded proteins, E1 and E2. Using a transient replication assay for human papillomavirus type 18 (HPV-18) DNA, we have found that two adjacent sequences present within the origin of replication can independently support replication. The first, a 77-bp region, contains one E2 binding site (E2BS) and a 16-bp inverted repeat element that probably corresponds to the E1 binding site (E1BS). The other, an 81-bp region, includes two E2BS but lacks the putative E1BS. A synthetic 33-bp oligonucleotide containing two high-affinity E2BS was also found to function as an origin of replication. Replication of all these plasmids was absolutely dependent on the presence of the HPV-18 E1 and E2 proteins. The HPV-1a E1 and E2 proteins were also found to support replication of a plasmid containing the complete HPV-18 origin but failed to replicate a plasmid containing two E2BS alone. Our results suggest that the E2 protein can target E1 to the origin through the formation of an E1-E2 complex which is likely to be involved the initiation of replication.

Base Sequence↗

PubMed1997

A novel cellular site-specific DNA-binding protein cooperates with the viral NS1 polypeptide to initiate parvovirus DNA replication.

Replication of linear single-stranded parvovirus DNA proceeds by a rolling-hairpin mechanism which generates long, palindromic, duplex concatamers. Processing to monomer length requires initiation from origins of DNA replication located at the 3' and 5' ends of each embedded monomer, reactions which can be recapitulated in vitro for minute virus of mice (MVM). To determine which cellular proteins were essential for replication from these origins, S100 extracts from 293S cells were fractionated on phosphocellulose. When recombined, these fractions were able to support replication in vitro, dependent on the viral initiator protein NS1, using plasmid forms of the 5' origin or the minimal 3' origin as templates. Fraction P-cell 1 contains two factors, replication protein A (RPA) and proliferating-cell nuclear antigen (PCNA), known to be essential for simian virus 40 replication in vitro. When P-cell 1 was replaced with purified recombinant RPA and PCNA, NS1-mediated MVM replication initiated from the 5' origin but not from the 3' origin. The 3' origin is a 50-bp sequence containing three distinct recognition elements, an NS1 binding site, a site at which NS1 nicks the DNA to generate the priming 3' OH, and a region containing a consensus activated transcription factor (ATF) binding site. To identify the missing factor(s) for 3' origin replication, P-cell 1 was fractionated by further chromatography and active fractions were identified by their ability to complement RPA, PCNA, and P-cell 2 for NS1-mediated, origin-specific replication. Gel shift and UV cross-linking analysis of the replication-competent fractions revealed a novel 110-kDa sequence-specific DNA binding protein which recognized the consensus ATF binding site region of the origin and which we have termed parvovirus initiation factor, or PIF. Binding of PIF appears to activate the endonuclease function of NS1, allowing efficient and specific nicking of the 3' minimal origin under stringent conditions in vitro.

Animals↗

PubMed1998

Role of the adenovirus DNA-binding protein in in vitro adeno-associated virus DNA replication.

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences.

Adenoviridae↗

PubMed1999

Interaction of the transcription factor TFIID with simian virus 40 (SV40) large T antigen interferes with replication of SV40 DNA in vitro.

Simian virus 40 (SV40) large tumor (T) antigen is the major regulatory protein that directs the course of viral infection, primarily by interacting with host cell proteins and modulating their functions. Initiation of viral DNA replication requires specific interactions of T antigen bound to the viral origin of DNA replication with cellular replication proteins. Transcription factors are thought to stimulate initiation of viral DNA replication, but the mechanism of stimulation is poorly understood. Since the transcription factor TATA-binding protein (TBP) binds to sequences within the origin of replication and interacts specifically with T antigen, we examined whether TBP complexes stimulate SV40 DNA replication in vitro. On the contrary, we found that depletion of TBP complexes from human cell extracts increased their ability to support viral DNA replication, and readdition of TBP complexes to the depleted extracts diminished their activity. We have mapped the sites of interaction between the proteins to residues 181 to 205 of T antigen and 184 to 220 of TBP. Titration of fusion proteins containing either of these peptides into undepleted cell extracts stimulated their replication activity, suggesting that they prevented the T antigen-TBP interaction that interfered with replication activity. TBP complexes also interfered with origin DNA unwinding by purified T antigen, and addition of either the T antigen or the TBP fusion peptide relieved the inhibition. These results suggest that TBP complexes associate with a T-antigen surface that is also required for origin DNA unwinding and viral DNA replication. We speculate that competition among cellular proteins for T antigen may play a role in regulating the course of viral infection.

Animals↗

PubMed2000

The E8E2C protein, a negative regulator of viral transcription and replication, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes.

The viral E2 protein is a major regulator of papillomavirus DNA replication. An important way to influence viral replication is through modulation of the activity of the E2 protein. This could occur through the action of truncated E2 proteins, called E2 repressors, whose role in the replication cycle of human papillomaviruses (HPVs) has not been determined. In this study, using cell lines that contain episomal copies of the "high-risk" HPV type 31 (HPV31), we have identified viral transcripts with a splice from nucleotide (nt) 1296 to 3295. These transcripts are similar to RNAs from other animal and human papillomaviruses and have the potential to fuse a small open reading frame (E8) to the C terminus of E2, resulting in an E8E2C fusion protein. E8E2C transcripts were present throughout the complete replication cycle of HPV31. A genetic analysis of E8E2C in the context of the HPV31 genome revealed that mutation of the single ATG of the E8 gene, introduction of a stop codon downstream of the ATG, or disruption of the splice donor site at nt 1296 led to a dramatic 30- to 40-fold increase in the transient DNA replication levels in both normal and immortalized human keratinocytes. High-level expression of E8E2C from heterologous vectors was found to inhibit E1-E2-dependent DNA replication of an HPV31 origin of replication construct as well as to interfere with E2's ability to transactivate reporter gene constructs. In addition, HPV31 E8E2C strongly repressed the basal activity of the major viral early promoter P97 independent of E2. E8E2C may therefore exert its negative effect on viral DNA replication through modulating E2's ability to enhance E1-dependent DNA replication as well as by regulating viral gene expression. Surprisingly, HPV31 genomes that were unable to express E8E2C could not be maintained extrachromosomally in human keratinocytes in long-term assays despite high transient DNA replication levels. This suggests that the E8E2C protein may play a role in copy number control as well as in the stable maintenance of HPV episomes.

Base Sequence↗