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PubMed2000

The immunogenicity and efficacy of replication-defective and replication-competent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle.

Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.

Adenoviridae↗

PubMed2003

Ad-mTERT-delta19, a conditional replication-competent adenovirus driven by the human telomerase promoter, selectively replicates in and elicits cytopathic effect in a cancer cell-specific manner.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, functions to stabilize telomere length during chromosomal replication. Previous studies have shown that hTERT promoter is highly active in most tumor and immortal cell lines but inactive in normal somatic cell types. The use of wild-type hTERT promoter, however, may be limited by its inability to direct high level and cancer cell-specific expression necessary for effective targeted gene therapy. To improve cancer cell specificity and the strength of the hTERT promoter, a modified hTERT, m-hTERT promoter was generated in which additional copies of c-Myc and Sp1 binding sites were incorporated adjacent to the promoter. As assessed using relative lacZ expression, hTERT and m-hTERT promoter activity was significantly upregulated in cancer cells but not in normal cells, and within these upregulated cancer cells, m-hTERT promoter strength was substantially higher than that of the wild-type hTERT. Next, to restrict viral replication to tumor cells, a conditional replication-competent adenoviruses, Ad-TERT-Delta19 and Ad-mTERT-delta19 were generated in which the E1A gene, which is essential for viral replication, was placed under the control of the hTERT and m-hTERT promoter, respectively. While the wild-type Ad-TERT-delta19 replicated in and induced cytopathic effect in cancer and in some normal cell lines, Ad-mTERT-delta19 enhanced viral replication and cytopathic effect only in cancer cells. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-mTERT-delta19. Taken together, present results strongly suggest that the use of the m-hTERT promoter is not only useful in the regulation of therapeutic gene expression but also that replication-competent oncolytic adenovirus under the control of the m-hTERT promoter may be a new promising tool for the treatment of human malignancies.

Adenoviridae↗

PubMed2025

Nanopore-based sequencing of active DNA replication reveals key principles of metazoan replication fork progression, origin and termination sites.

Balancing replication fork progression and origin usage is essential to maintain genome stability, but measuring replication fork progression rates and origin usage throughout the genome has been challenging. Here, we use nanopore sequencing combined with DNAscent to measure replication fork progression together with origin and termination site usage with single-molecule precision throughout the Drosophila genome with nearly full genome coverage. We find that replication fork progression rates are not uniform throughout the genome. Rather, fork progression is slowest in euchromatin, and this is not correlated with active transcription. Replication origins are also influenced by chromatin, but the exact position of initiation is highly variable and are often several kilobases away from ORC binding sites. Termination sites lack any chromatin or sequence motifs and appear nearly random throughout the genome. By measuring DNA replication dynamics at near full genome coverage, our work reveals key principles of metazoan replication dynamics.

Journal Article↗

PubMed2000

Mutation analysis of replicative genes encoding the large subunits of DNA polymerase alpha and replication factors A and C in human sporadic colorectal cancers.

We examined cDNAs of the catalytic subunit of DNA polymerase alpha (185 kDa), the 70 kDa subunit of replication protein A (single-stranded DNA-binding protein) and the 140 kDa subunit of replication factor C for mutations. Surgical specimens from 12 patients with sporadic colon cancer and normal mucosae from the same patients were investigated. In addition, we analyzed 3 human colon cancer cell lines that exhibited defects in mismatch repair (DLD-1, HCT116, SW48) and 3 colon cancer cell lines without such a defect (HT29, SW480 and SW620). For detection of mutations, we used reverse transcription of mRNA, amplification of cDNAs by PCR, analysis of single-strand conformation polymorphism and DNA sequencing. Eleven colon cancers and 6 colon cancer cell lines were analyzed for DNA polymerase alpha. Only 2 silent point mutations were detected, in 1 colon carcinoma and in cell line HCT116. Two sequence alterations of the 70 kDa subunit of replication factor A were identified in 15 specimens (9 colon carcinomas and 6 cell lines). Colon carcinomas from 2 patients (CC5MA and CC25HN) exhibited an ACA-->GCA transition in codon 351, which caused a Thr-->Ala exchange. In carcinomas CC5MA and CC8MA, a TCC-->TCT (Ser-->Ser) transition in codon 352 was observed. The deviations in codons 351 and 352 occurred in both cancer tissues and normal mucosae, suggesting a genetic polymorphism. No mutation was found in the 140 kDa subunit of replication factor C from 16 specimens (10 tumors and 6 cell lines). Point mutations were identified in the p53 tumor-suppressor gene in 4 of the 6 colon cancer cell lines and 3 of the 8 carcinoma specimens. We did not find tumor-associated DNA sequence alterations that resulted in amino acid changes in the DNA replication genes analyzed. We infer that the scarcity of mutations found is due to stringent selection, eliminating functionally impaired replication proteins.

Colorectal Neoplasms↗

PubMed1995

Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication.

Geminiviruses are plant DNA viruses with small genomes whose replication, except for the viral replication protein (Rep), depends on host proteins and, in this respect, are analogous to animal DNA tumor viruses, e.g. SV40. The mechanism by which these animal viruses create a cellular environment permissive for viral DNA replication involves the binding of a virally encoded oncoprotein, through its LXCXE motif, to the retinoblastoma protein (Rb). We have identified such a LXCXE motif in the Rep protein of wheat dwarf geminivirus (WDV) and we show its functional importance during viral DNA replication. Using a yeast two-hybrid system we have demonstrated that WDV Rep forms stable complexes with p130Rbr2, a member of the Rb family of proteins, and single amino acid changes within the LXCXE motif abolish the ability of WDV Rep to bind to p130Rbr2. The LXCXE motif is conserved in other members of the same geminivirus subgroup. The presence of an intact Rb binding motif is required for efficient WDV DNA replication in cultured wheat cells, strongly suggesting that one of the functions of WDV Rep may be the linking between viral and cellular DNA replication cycles. Our results point to the existence of a Rb-like protein(s) in plant cells playing regulatory roles during the cell cycle.

Amino Acid Sequence↗

PubMed1999

DNA replication errors produced by the replicative apparatus of Escherichia coli.

It has been hard to detect forward mutations generated during DNA synthesis in vitro by replicative DNA polymerases, because of their extremely high fidelity and a high background level of pre-existing mutations in the single-stranded template DNA used. Using the oriC plasmid DNA replication in vitro system and the rpsL forward mutation assay, we examined the fidelity of DNA replication catalyzed by the replicative apparatus of Escherichia coli. Upon DNA synthesis by the fully reconstituted system, the frequency of rpsL-mutations in the product DNA was increased to 1.9x10(-4), 50-fold higher than the background level of the template DNA. Among the mutations generated in vitro, single-base frameshifts predominated and occurred with a pattern similar to those induced in mismatch-repair deficient E. coli cells, indicating that the major replication error was slippage at runs of the same nucleotide. Large deletions and other structural alterations of DNA appeared to be induced also during the action of the replicative apparatus.

Base Pairing↗

PubMed1986

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

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

Cross-Linking Reagents↗

PubMed1989

The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication.

Epstein-Barr virus (EBV) oriP contains two components, a dyad symmetry element and a direct repeat element, that, in the presence of EBV nuclear antigen 1, are necessary and sufficient for plasmid replication. We have examined the replicative forms generated by EBV oriP using 2D gel electrophoresis. The patterns obtained from an oriP plasmid in a transfected cell line indicate that the site of initiation of DNA replication is at or very near the dyad symmetry element, while the direct repeats contain a replication fork barrier and the termination site. Thus, replication from oriP proceeds in a predominantly undirectional manner. The patterns obtained from cells immortalized by EBV suggest that replication from oriP proceeds similarly in the viral genome.

DNA Replication↗

PubMed2003

Regulation of alternative replication bypass pathways at stalled replication forks and its effects on genome stability: a yeast model.

Replication-blocking lesions result in increased genomic instability by stalling replication forks. Eukaryotic cells appear to have evolved several surveillance and repair/bypass mechanisms to ensure that replication can be resumed at these stalled forks. In the yeast Saccharomyces cerevisiae, the helicases Srs2 and Sgs1 appear to play a role in controlling the processing and stabilization of stalled replication forks. These proteins appear to be tightly regulated throughout the cell cycle and play a direct role in DNA-damage checkpoints. This allows the cells to determine the best mechanism to reestablish replication at the stalled fork: by shuttling the lesion into the RAD6-dependent pathway that can lead to error-free or error-prone bypass; or by using homologous recombination. Under conditions where both the RAD6-dependent pathway and recombination are disabled, the cells can bypass the lesion using a novel damage avoidance mechanism that is controlled by Mgs1. Replication fork bypass processes appear to be highly conserved within eukaryotes, with homologs for SGS1 and MGS1 found in both Schizosaccharomyces pombe and mammalian cells.

Crossing Over, Genetic↗

PubMed2003

Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA.

The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli

Bacterial Proteins↗

PubMed2000

Initiation of genome replication: assembly and disassembly of replication-competent chromatin.

Considerable progress has been made in research on the initiation of eukaryotic genome replication. This has generated a number of recent review articles. Here, we briefly summarize the major conclusions described in these articles and also include the results of more recent primary articles. The consensus view that has emerged is that a pre-replication complex assembles during the G1 phase of the cell cycle, making chromatin competent for replication. The complex consists of Orc proteins, Cdc6p, and the family of Mcm proteins. Chromatin, thus 'licenced' for replication, is guided into the S phase by the activation of cell-cycle-regulated protein kinases. Upon entry into S phase, the pre-replication complex is partially dissolved, first by the dissociation of Cdc6p and then by the gradual release of Mcm proteins. This appears to be accompanied by a recruitment of chain elongation factors and the establishment of replication forks.

Animals↗

PubMed2001

Bacteriophage T4 proteins replicate plasmids with a preformed R loop at the T4 ori(uvsY) replication origin in vitro.

Bacteriophage T4 DNA replication proteins catalyze complete unidirectional replication of plasmids containing the T4 ori(uvsY) replication origin in vitro, beginning with a preformed R loop at the position of the origin R loop previously identified in vivo. T4 DNA polymerase, clamp, clamp loader, and 32 protein are needed for initial elongation of the RNA, which serves as the leading-strand primer. Normal replication is dependent on T4 41 helicase and 61 primase and is strongly stimulated by the 59 helicase loading protein. 59 protein slows replication without the helicase. As expected, leading-strand synthesis stalls prematurely in the absence of T4 DNA topoisomerase. A DNA unwinding element (DUE) is essential for replication, but the ori(uvsY) DUE can be replaced by other DUE sequences.

Bacteriophage T4↗

PubMed2001

DNA damage by the enediyne C-1027 results in the inhibition of DNA replication by loss of replication protein A function and activation of DNA-dependent protein kinase.

Treatment of cells with the enediyne C-1027 is highly efficient at inducing single- and double-strand DNA breaks. This agent is highly cytotoxic when used at picomolar levels over a period of days. For this study, C-1027 has been used at higher levels for a much shorter time period to look at early cellular responses to DNA strand breaks. Extracts from cells treated with C-1027 for as little as 2 h are deficient in SV40 DNA replication activity. Treatment with low levels of C-1027 (1-3 nM) does not result in the presence of a replication inhibitor in cell extracts, but they are deficient in replication protein A (RPA) function. Extracts from cells treated with high levels of C-1027 (10 nM) do show the presence of a trans-acting inhibitor of DNA replication. The deficiency in RPA in extracts from cells treated with low levels of C-1027 can be fully complemented by the addition of exogenous RPA, and may be due to a C-1027-induced decrease in the extractability of RPA. This decrease in the extractability of RPA correlates with the appearance of many extraction-resistant intranuclear RPA foci. The trans-acting inhibitor of DNA replication induced by treatment of cells with high levels of C-1027 (10 nM) is DNA-dependent protein kinase (DNA-PK). DNA-PK is activated by the presence of DNA fragments induced by C-1027 treatment, and can be abrogated by removal of the DNA fragments. Although it is activated by DNA damage and phosphorylates RPA, DNA-PK is not required for either RPA focalization or loss of RPA replication activity.

Aminoglycosides↗

PubMed1996

DNA replication machinery: functional characterization of a complex containing DNA polymerase alpha, DNA polymerase delta, and replication factor C suggests an asymmetric DNA polymerase dimer.

By using a complementation assay for a replication factor C dependent DNA polymerase activity on a singly-primed M13 DNA template, we have isolated from calf thymus a multiprotein complex active in DNA replication. For this, the inclusion of ATP during the entire isolation procedure was essential, since the complex decayed after omission of ATP. This complex contains at least DNA polymerase alpha/primase, DNA polymerase delta, and replication factor C as shown by gel-filtration and coimmunoprecipitation experiments. It is functionally active in replication of primed and unprimed single-stranded M13 DNA templates. Furthermore, in the presence of proliferating cell nuclear antigen and ATP, it forms an isolatable holoenzyme/template-primer complex. Replication factor C apparently mediates the interaction of DNA polymerase delta in the complex with proliferating cell nuclear antigen, through an ATP-dependent mechanism. This interaction appears to stabilize the binding of the complex to a template-primer and to coordinate the activity of DNA polymerase alpha/primase and DNA polymerase delta during replication of a single-stranded DNA template. Our data suggest the existence of an asymmetric DNA polymerase complex in mammalian cells.

Adenosine Triphosphate↗

PubMed2000

Mid-cell Z ring assembly in the absence of entry into the elongation phase of the round of replication in bacteria: co-ordinating chromosome replication with cell division.

We have shown previously that, when spores of a thymine-requiring strain of Bacillus subtilis were grown out in the absence of thymine, mid-cell Z rings formed over the nucleoid and much earlier than might be expected with respect to progression into the round of replication. It is now shown that such conditions allow no replication of oriC. Rather than replication, partial degradation of the oriC region occurs, suggesting that the status of this region is connected with the 'premature' mid-cell Z ring assembly. A correlation was observed between entry into the replication elongation phase and a block to mid-cell Z rings. The conformation of the nucleoid under various conditions of DNA replication inhibition or limitation suggests that relief of nucleoid occlusion is not primarily responsible for mid-cell Z ring formation in the absence of thymine. We propose the existence of a specific structure at mid-cell that defines the Z ring nucleation site (NS). It is suggested that this NS is normally masked by the replisome upon initiation of replication or soon after entry into the elongation phase, and subsequently unmasked relatively late in the round. During spore outgrowth in the absence of thymine, this checkpoint control over mid-cell Z ring assembly breaks down prematurely.

Bacillus subtilis↗

PubMed2000

Replication-induced transcription of an autorepressed gene: the replication initiator gene of plasmid P1.

The replication origin of plasmid P1 contains an array of five repeats (iterons) that bind the plasmid-encoded initiator RepA. Within the array lies the repA promoter, which becomes largely repressed on RepA binding (autorepression). One might expect that extra iterons produced on plasmid replication would titrate RepA and release the repression. The promoter, however, is induced poorly by extra iterons. The P1 copy number is reduced by extra iterons in the presence of the autorepressed repA gene but not when additional RepA is provided from constitutive sources. It has been proposed that the iteron-bound RepA couples with the promoter-bound RepA and thereby maintains repression. Although not the product of replication, we find that the act of replication itself can renew RepA synthesis. Replication apparently cleans the promoter of bound RepA and provides a window of opportunity for repA transcription. We propose that replication-induced transcription is required to ensure initiator availability in a system that is induced poorly when challenged with additional initiator binding sites.

DNA Helicases↗

PubMed1999

Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication.

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.

Cloning, Molecular↗

PubMed1995

The role of the 34-kDa subunit of human replication protein A in simian virus 40 DNA replication in vitro.

Human replication protein A (RPA) is a three subunit protein complex involved in DNA replication, repair, and recombination. We investigated the role of the 34-kDa subunit (p34) of RPA in DNA replication by generating a series of p34 mutants. While deletion of the N-terminal domain of p34 prevented its phosphorylation by both cyclin-dependent kinase (Cdk) and DNA-dependent kinase, a double point mutant that lacks the major phosphorylation sites for Cdk could be phosphorylated by DNA-dependent kinase. In simian virus 40 (SV40) DNA replication, RPA containing either of these mutants functioned as efficiently as wild-type RPA. However, mutant RPA containing C-terminally deleted p34 was only marginally active. This indicates that the C-terminal region, but not the phosphorylation domain of p34, is necessary for RPA function in DNA replication. Furthermore, RPA containing the C-terminally deleted p34 mutant could stimulate DNA polymerase alpha, and bind to single-stranded DNAs but was limited in its ability to unwind DNA or interact with SV40 large T antigen (T Ag). These results suggest that RPA p34 interacts with SV40 T Ag during the initiation of SV40 DNA replication and may be necessary for DNA unwinding.

Base Sequence↗